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3rd INTERNATIONAL SYMPOSIUM ON TRACE ELEMENTS

AND HEALTH

3rd INTERNATIONAL SYMPOSIUM ON TRACE

ELEMENTS AND HEALTH

Murcia, Spain, 24-27 May, 2011

I.S.B.N.: 978-84-694-3922-7Depósito Legal: MU 626-2011Fotocomposición e impresión: COMPOBELL, S.L.

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Summary

Welcome letter .............................................................................. 9

Honour Committees .................................................................... 11

Location. About Murcia .............................................................. 15

Scientific Programme for TRACEL 2011 and Cost FA0905 Conference Murcia, Spain ..................................................... 19

Session I: Trace elements in livestock ........................................ 41

Session II: Trace elements as food fortificants .......................... 55

Session III: Toxicology and risk assessment of trace elements 61

Session IV: Bioavailability of trace elements ............................ 79

Session V: Trace elements and health ........................................ 93

Session VI: Biofortification of trace elements (Joint session with Cost Action FA0905) ........................................................... 103

Summary

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Session VII: Trace element analysis and quality control ........ 125

Session VIII (Cost action FA0905) homeostasis of trace elements in plants ......................................................................................... 141

Posters ............................................................................................ 155

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Welcome letter

Dear colleagues:

On behalf of the Organizing Committee it is a great pleasure that I invite you to attend the 3rd International Symposium on Trace Elements and Health to be held in Murcia, Spain, 25- 27 May, 2011.

The aim of this interdisciplinary meeting is to gather experts with different backgrounds to discuss all aspects of trace elements in relation to soil, crop production, animal husbandry, food, nutrition and human health as one related subject. Given that diet is the main source of trace elements, the production of nutritious and safe food both sufficiently and sustainably is the ultimate goal of modern agriculture. At the same time, the presence of trace elements in animal feed, either as contaminants or as additives aimed to improve animal health or food properties. However, closer coordination is needed between the nutrition and toxicology communities to set acceptable ranges of intake for trace elements, taking into account its bioavailability in foods and genetic and individual differences in the targeted population.

Welcome letter

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This meeting follows two previous ones, organized in Hangzhou, China (2004) and Helsinki, Finland (2007). As the two previous meetings of this series, the symposium aims to be a suitable forum for new ideas and experiences which can be shared among researches working on trace elements in agroecosystems, animal and human health.

We trust that you will have a fruitful meeting and a very pleasant stay in Murcia, a city where you will find everything without forgetting its sunny weather, wonderful beaches, high quality gastronomy, historical Roman and Arabian ruins, charming people…

With kind regards,

Carmen Martínez GraciáSymposium Chair

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Honour Committees

· Excmo. Sr. D. Ramón Luis Valcárcel Siso. Presidente de la Comunidad Autónoma de la Región de Murcia.

· Excma. Sra. Dª María Ángeles Palacios Sánchez. Consejera de Sanidad y Consumo.

· Ilmo. Sr. D. Miguel Ángel Cámara Botía. Alcalde del Excmo. Ayuntamiento de Murcia.

· Excmo. Sr. D. José Antonio Cobacho Gómez. Rector Magnífico de la Universidad de Murcia.

· Ilmo. Sr. D. Antonio José Rouco Yáñez. Decano de la Facultad de Veterinaria de la Universidad de Murcia.

Organizing Committee TRACEL2011

· Carmen Martínez Graciá, Chair, Department of Food Science and Nutrition, University of Murcia.

· Gaspar Ros Berruezo, Department of Food Science and Nutrition, University of Murcia.

· María Jesús Periago Castón, Department of Food Science and Nutrition, University of Murcia.

Honour Committees

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· Carmen Frontela Saseta, Department of Food Science and Nutrition, University of Murcia.

· Darío Pérez Conesa, Department of Food Science and Nutrition, University of Murcia.

· Francisco Javier García Alonso, Department of Food Science and Nutrition, University of Murcia.

· Marina Santaella Pascual, Department of Food Science and Nutrition, University of Murcia.

· Patricia Peso Echarri, Department of Food Science and Nutrition, University of Murcia.

· Eduardo Pérez Asensio, Department of Food Science and Nutrition, University of Murcia.

· Miguel Motas Guzmán, Department of Health Sciences, University of Murcia.

· Rosaura Farré Rovira, Food Science and Nutrition, University of Valencia.

Cost Action FA0905: Mineral-improved crop production for healthy food and feed

· Prof. Bal Ram Singh, COST Action FA0905 Chairmen, Norwegian University of Life Sciences (UMB), Ås, Norway.

· WG2: Co-ordinators: Prof. Nathalie Verbruggen, Belgium. Assoc. Prof. Mark, G.M. Arts, Netherlands. Prof. Danuta Maria Antosiewicz, Poland.

· WG4: Co-ordinators: Prof. Richard Hurrell, Switzerland. Dr. Martin Broadley, UK.

Honour Committees

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International Scientific Committee

· Bal Ram Singh, Norwegian University of Life Sciences, Norway.

· Björn Akesson, Lund University, Sweden.· Glenn B. Gregorio, Africa Rice Center (WARDA), Nigeria.· Holger Kirchman, Swedish University of Agricultural Science,

Sweden.· Ismail Cakmak, Sabanci University, Turkey.· Jonathan Wong, Hong Kong Baptist University, Hong Kong,

China.· Manfred Anke, Friedrich Schiller University of Jena, Germany.· Margaret Raymann, University of Surrey, UK.· Masajaru Murakami, National Institue for Agro-environmental

Sciences, Japan.· Neville F. Suttle, Moredun Research Institute, UK.· Richard H. Loeppert, Texas A&M University, USA.· Rorhieh Hajiboland, University of Tabriz, Iran.· Staffan Skerfving, Lund University of Life Sciences, Norway.· Xiaoe Yang, Zhejiang University, China.· Zhenli He, University of Florida, USA.· Yusuf Genc, Molecular Plant Breeding CRC, Australia.· Zed Rengel, University of Western Australia, Australia.

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Location - About Murcia

The city of Murcia was founded in 831 by Abd-Al-Rahman II as part of Cordoba, on a privileged location, in the centre of the Valley of the Segura River. The city wall offers a good idea of the importance achieved by the city under Arab rule, and the ruins of part of the wall are still visible in different places around the city.

Location. About Murcia

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Finished in the 15th century, the Baroque Cathedral is the most impressive monument in Murcia. The Cathedral is flanked by other eighteenth century buildings - the Episcopal Palace and the old Granary, known as El Almudí, both good examples of the construction boom that took place during the above mentioned period. The streets and squares of Murcia also offer magnificent examples of 19th century architecture, such as the Town Hall, the Victoria Hotel, the Romea Theatre and particularly the Casino, built in 1847.

However, Murcia, rather than remain anchored in its past, has developed into a modern city, equipped with all the necessary facilities and infrastructures. The mild climate with very little


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precipitation and the diversified ecological landscape draws you into an incredible state of well being. Its coasts, with over 200 beaches, shares two different bodies of water: the Mediterranean and the Menor Sea, which has an outstretched piece of land called La Manga.

Cartagena is the second largest town in the Region of Murcia, it was founded by the Carthaginians in 223 BC. The Romans, Byzantines and Arabs all took full advantage of Cartagena’s perfect natural harbour, surrounded by mountains that were rich in metallic ores. Today this natural harbour is still the home of the biggest Spanish naval base in the Mediterranean.


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SCIENTIFIC PROGRAMME FOR TRACEL 2011 AND COST FA0905

CONFERENCE MURCIA, SPAIN

1. Tuesday 24th May

19:00-22:00: Welcome reception.

Venue: Claustro de la Facultad de Derecho, Campus de la Merced, Murcia.

2. Wednesday 25th May

Venue: Aula Obra Social Caja Murcia. Gran Vía. Murcia.

8:00-9:00: Registration and Mounting of Posters.

9:00-9:30: Official Opening.

Scientific Programme for Tracel 2011 and Cost FA0905 Conference Curcia, Spain

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SESSION I: TRACE ELEMENTS IN LIVESTOCK

Chair: Dr. Neville Suttle. Moredun Foundation, Penicuik, UK.

Rapporteur: Dr. Botha C. University of Pretoria, South Africa.

9:30-10:10: Plenary Conference: “Trace Elements and Animal Health: What is new?”

Keynote Speaker: Dr. Jerry W. Spears. Department of Animal Science and Interdepartamental Nutrition Program. North Carolina State University, Raleigh, U.S.A.

10:10-10:30: Short Communication: “Interactions between gut infections and copper-induced hepatotoxicity in ruminants: possible mechanisms and implications”. Dr. Neville Suttle. Moredun Foundation, Penicuik, UK.

10:30-10.45: Oral presentation: “Impact of EU legislation on trace element and mineral supplementation of livestock”. Packington, A., Pickard, D. DSM Nutritional Products Ltd. Derbyshire, UK.

10:45-11:00: Oral presentation: “Iron and Zinc content in meat from cattle with chronic fascioliasis”. Sánchez, P., Ros, G., Martínez Graciá, C. Faculty of Veterinary Science, University of Murcia. Spain.

11:00-11:30: Coffee Break.

11:30-11:45: Oral presentation: “Excess iodine intake by the ewe in late pregnancy reduces immunoglobulin G absorption in the newborn lamb”. Boland, T.M. School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Ireland.


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11:45-12:00: Oral presentation: “Veterinary and public health implications of cattle farmed in the vacinity of a vanadium processing plant”. Botha, C.J., Gummow, B. Faculty of Veterinary Science, University of Pretoria, South Africa.

SESSION II: TRACE ELEMENTS AS FOOD FORTIFICANTS

Chair: Dr. Sue Fairweather-Tait. Norwich Medical School, University of East Anglia, Norwich, Norfolk, UK.

Rapporteur: Dr. Zhenli He. Institute of Food and Agricultural Sciences, University of Florida, USA.

12:15-12:50: Plenary Conference: “New Developments in Food Fortification”.

Keynote Speaker: Dr. Michael Zimmermann. Human Nutrition Laboratory, Institute of Food, Nutrition and Health, ETH Zurich, Switzerland.

12:50-13:10: Short Communication: “Application of nanotechnology in Agriculture and Health: Progress and perspectives”. Dr. Zhenli He. Yang, X.E., Bakshi, S., Stofella, P.J. Indican River Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, USA.

13:10-13:30: Poster viewing.

13:30-15:30: Lunch (NH Amistad Hotel).


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SESSION III: TOXICOLOGY AND RISK ASSESSMENT OF TRACE ELEMENTS

Chair: Dr. Xiaoe Yang, Zheijiang University, China.

Rapporteur: Dr. Miguel Motas. University of Murcia, Spain.

15:30-16:10: Plenary Conference: “Movement of Heavy metals from soil to edible parts of crops (Human food Chain) and risk Assessment”.

Keynote speaker: Dr. Xiaoe Yang, He, Z.L., Xiao, W., Tarig, M. MOE Key Labortatory of Environmental Remediation and Ecological Health, College of Environmental and Resource Sciences, Zheijiang University, China.

16:10-16:30: Short Communication: “Toxicology and trace metals risk assessment: a total diet study of the Spanish population”. Carmen Rubio Armendáriz, Toxicology Department, La Laguna University, Tenerife, España.

16:30-16:45: Oral presentation: “Presence and distribution of trace elements and heavy metals in adult antarctic penguins: background levels?” Jerez, S., Motas, M., Palacios, M.J., Cuervo, J.J., Díaz, J., Benzal, J., Vidal, V., Barbosa, A. Toxicology Area, Veterinary Faculty, University of Murcia, Spain.

16:45- 17:15: Coffe Break.

17:15- 17:30: Oral presentation: “Renal effects of low environmental exposure to cadmium: evidence of reverse causation”. Bernard, A., Chaumont, A. Unit of Toxicology and Applied Pharmacology, Catholic University of Louvain, Brussels, Belgium.


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17:30-17:45: Oral presentation: “Cadmium intake from bean plants grown in tropical contaminated soils”. Nogueira, TAR., Abreu Junior, CH., He ZL., Alleoni, LRF., Trombeta, ALB., Malavolta, E. University of Sao Paulo (CENA/USP), Piracicaba SP, Brazil.

17:45-18:00: Oral presentation: “Relation of fish consumption to mercury, selenium and arsenic levels in cord blood and breast milk from Slovenian pregnant women”. Miklavcic A., Mazej, D., Tratnik, J., Kobal, A.B., Krsnik, M., Planinsek, P., Osredkar, J., Horvat, M. Jozef Stefan Institute, Department of Environmental Sciences, Ljubljana, Slovenia.

18:00-18:15: Oral presentation: “Evaluation of the assimilation of heavy metals by plants in contaminated soils submitted to a remediation process”. Martínez-Sánchez, M.J., Pérez-Sirvent, C., Cánovas, J., Agudo, I. Banegas, A. Department of Agricultural Chemistry, Geology and Pedology. University of Murcia. Spain.

19:00: Social event: Tour around the city of Murcia.

3. Thursday 26th May


SESSION IV: BIOAVAILABILITY OF TRACE ELEMENTS

Chair: Dr. Rosaura Farré. University of Valencia. Spain.

Rapporteur: Dr. Rachel Hurst. University of East Anglia UK.


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9:00-9:40: Plenary Conference: “Bioavailability of iron from whole diets in adults”.

Keynote speaker: Dr. Susan J. Fairweather-Tait, Harvey, L.J., Collings, R., Casgrain, A., Hurst, R., Hooper, L. Norwich Medical School, University of East Anglia, Norwich, Norfolk, UK.

9:40-9:55: Oral presentation: “Selenium uptake, accesibility and bioavailability in wheat”. Govasmark, E., Brant-Kjelsen, A., Salbu, B. Norwegian University of Life Sciences. Department of Plant and Environmental Science, Norway.

9:55-10:10: Oral presentation: “Effect of Bifidobacterium phytases on in vitro mineral availability from infant cereals”. Sanz Penella, J.M., Frontela, C., Ros, G., Martínez Graciá, C., Monedero, V., and Haros, M. Institute of Agrochemistry and Food Technology, (IATA-CSIC), Valencia, Spain.

10:10-10:25: Oral presentation: “Effect of processing and food matrix on calcium and phosphorus bioavailability from milk-based fruit beverages in Caco-2 Cells”. Cilla, A., Lagarda, M.J., Alegría, A., Sánchez-Moreno, C., Plaza, L., Cano M.P., De Ancos, B., Barberá R. Department of Nutrition and Food Chemistry. Faculty of Pharmacy. University of Valencia, Spain.

10:25-10:40: Oral presentation: “Uptake and transport of calcium, iron and zinc from an infant formula supplemented with alpha-lactalbumin and nucleotides, using Caco-2 Cells”. Peso Echarri, P., Frontela Saseta, C., González Bermúdez, C., González Marín, A., Ros Berruezo, G., Martínez Graciá, C. Department of Food Science and Nutrition, Faculty of Veterinary Science, University of Murcia, Spain. COST FA 0905.


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10:40-10:55: Oral presentation: “Iron, calcium and zinc in vitro solubility and availability from different commercial infant formulas with thickening agents”. González Bermúdez, C.A., Frontela Saseta, C., Peso Echarri, P., López Nicolás R., Ros Berruezo, G., Bernal Cava M.J., Martínez Graciá, C. Department of Food Science and Nutrition, Faculty of Veterinary Science, University of Murcia, Spain. COST FA 0905.


SESSION V: TRACE ELEMENTS AND HEALTH

Chair: Dr. Martin Broadley. University of Nottingham, UK.

Rapporteur: María Hermoso, Munich Medical Centre, Germany.

11:30-12:10: Plenary Conference: “Dose-Response meta-analysis to elucidate the relationship between plasma selenium and prostate cancer risk”.

Keynote speaker: Dr. Rachel Hurst., Hooper, L., Norat, T., Lau, R., Aune, D., Greenwood, D., Collings, R., Fairweather-Tait, S.J. Norwich Medical School, University of East Anglia, Norwich, Norfolk, UK.

12:10-12:30: Short Communication: “Iron and neurodevelopment and immunity in infants, children and adolescents. A systematic review”- Dr. María Hermoso, Vucic, V., Volhardt, C., Arsic, A., Roman-Viñas, B., Iris Iglesia-Altaba, Gurinovic, M., Koletzko, B. Division of Metabolic and Nutritional Medicine. Dr. Von Hauner Children´s Hospital, Ludwig-Maximilians-University of Munich Medical Centre, Germany.


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12.30-12:45: Oral presentation: “Influence of an iron fortified, fruit juice on bone remodeling in women at risk of iron deficiency anaemia.” Blanco Rojo, R., Pérez Granados, A.M., De la Piedra, C., Vaquero M.P. Department of Metabolism and Nutrition, Institute of Food Science and Technology and Nutrition (ICTAN, CSIC), Madrid, Spain.

12:45-13:00: Oral presentation: “Elucidation of the role of zinc, zinc-enzyme and tetrahydrobiopterin in the alteration of catecholamine metabolism in the cerebral cortex with aging in senescence-accelerated mouse”. Miyajima, M., Minoshima, M., Tanaka, M., Nishimura, R., Hosokawa, T., Kurasaki, M., Saito, T. Graduate School of Health Sciences, Hokkaido University, Sapporo, Japan.


13:30-15:00: Lunch (Hotel NH Amistad).

SESSION VI: BIOFORTIFICATION OF TRACE ELEMENTS (Joint session with COST Action FA0905)

Chair: Prof. Bal Ram Singh. Norwegian University of Life Sciences. Norway.

Rapporteur: M.M Seppänen. University of Helsinki, Finland.

15:00-15:40: Plenary Conference: “Biofortification of foods with macro-and micro-mineral elements”.

Keynote speaker: Dr. Martin R. Broadley. Plant and Crop Sciences Division, School of Biosciences, University of Nottingham, UK.


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15:40- 16:15: Plenary Conference: “Zinc and iron biofortification: HarvestPlus Crop Development Update”.

Keynote speaker: Dr. Erick Boy. HarvestPlus, Washington DC, U.S.A.

16:15-16:30: Oral presentation: “Nutritional quality improvement in common beans by genetic reduction of phytates and other antinutritional factors”. Perrone, D., Doria, E., Tava, A., Dani V., Sparvoli, F, Glahn, R., Nielsen E., and Campion B. Dipartimento di Genetica e Microbiologia. Universitá di Pavia; CRA- Unitá di Ricerca per l’Orticoltura, Montanaso Lombardo, Lodi, Italy. COST FA 0905.

16:30-16-45: Oral presentation: “A defective ABC transporter of the MRP family, responsible of the bean low phytic acid (lpa1) mutation, affects the regulation of phytic acid pathway, reduces seed myo-inositol and alters ABC sensitivity.” Panzeri, D., Cassani, E., Doria, E., Tagliabue, G., Forti, L., Campion, B., Bollini, R., Brearley, C.A., Pilu, R., Nielsen, E., Sparvoli, F. Istituto di Biologia e Biotecnologia Agraria, CNR, Milano, Italy. COST FA 0905.


17:15-17:30: Oral presentation: “Model studies on Zn-biofortification: modification of Zn subcellular and tissue distribution through heterologous expression of HMA4 likely contributes to the generation of the phenotype”. Siemianowski, O. and Antosiewicz, D.M. University of Warsaw, Faculty of Biology, Institute of Plant Experimental Biology, Poland. COST FA 0905.

17:30-17:45: Oral presentation: “Size exclusion chromatography, coupled with inductively coupled plasma-mass spectrometry is an effective method of screening cereals for potentially bioavailable genotypes to aid future breeding strategies in


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tackling iron deficiency”. Eaglin T., Zhao, F., Neal, A., McGrath, S., Shewry, P., Fairweather-Tait, S.J. Rothamsted Research Hertfordshire, UK. COST FA 0905.

17:45-18:00: Oral presentation: “Determination of heritability of zinc-efficiency in pepper (Capiscum annum L.) and development of molecular markers linked to QTL/genes controlling zinc-efficiency.” Mutlu, N., Keles, D., Pinar, H., Ata, A. Akdeniz University, Faculty of Agriculture, Department of Agricultural Biotechnology, Antalaya, Turkey. COST FA 0905.

18:00-18:15: Oral presentation: “Effects of foliar zinc fertilizer application on zinc density and bioavailability in rice (Oriza Sativa, L.)”. Wei, Y., Shohag, M.J.I., Yang, X., Wang, Y., He, Z. Stoffella, P.J. MOE Key Laboratory of Environmental Remediation and Ecosystem Health, College of Environmental and Resources Science, Zhejiang University, China.


20:30: Symposium dinner.

4. Friday 27th May


SESSION VII: TRACE ELEMENT ANALYSIS AND QUALITY CONTROL

Chair: Prof. Peter Schröder. Helmholtz Zentrum München German Research Center for Environmental Health, Germany.


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Rapporteur: Asso.Prof. Mark Aarts. Wageningen University.

9:00-9:40: Plenary Conference: “Isotopic tools and metrology principles for reliable inorganic chemical measurements – illustration with reference values for the EU Reference Laboratory on heavy metals in feed and food”.

Keynote Speaker: Dr. Christophe R Quetel. European Commission. Joint Research Center. Institute for Reference Materials and Measurements.

9:40-10:00: Short Communication: “Ultra-trace speciation by online preconcentration Liquid Chromatography Inductively Coupled Plasma Mass Spectrometry to study the environmental fate of elemental selenium”. Lenz, M. Floor, G.H., Evangelou, M.W.J., Schulin, R., Corvini Philippe F.X. Institute for Ecopreneurship, University of Applied Sciences Northwestern Switzerland (FHNW), Switzerland. COST FA 0905.

10:00-10:15: Oral presentation: “Capillary Electrophoresis coupled with and Inductively Coupled Plasma mass Spectrometry detector permitted speciation and quantification of metal glycinate complexes in feed premixes”. Ionescu, C., Vacchina, V., Lobinski, R., Bravo, D. Pancosma, Geneva Switzerland.

10:15-10:30: Oral presentation: “Gas Chromatography coupled with atomic emission of mass spectrometry detectors for the determination of organotin compounds in foods”. Campillo, N. Viñas, P., Peñalver, R., Cacho, J.I., Hernández-Córdoba, M. Department of Analytical Chemistry, Faculty of Chemistry, University of Murcia. Spain.

10:30-11:00: Coffe break.


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11:00-12:00 Closing Lecture: “A diet for health and longevity. How do we get there?”.

Chair: Dr. Francisco Tomás Barberán. CEBAS (CSIC), Murcia. Spain.

Keynote speaker: Dr. Bruce N. Ames. Nutrition and Metabolism Center. Children´s Hospital Oakland Research Institute, California. USA.

12:00-12:30: Main conclusions. Closing of the Meeting.

Dr. Susan J. Fairweather-Tait

Dr. Xiaoe Yang

Dr. Carmen Martínez Graciá

16:00: Social Event: Tour to Cartagena (Optional).

SESSION VIII (COST ACTION FA 0905) HOMEOSTASIS OF TRACE ELEMENTS IN PLANTS

Chair: Prof. N. Verbruggen. Université Libre de Bruxelles.

Rapporteur: Dr. M. Mench. University of Bordeaux.

14:30-15:10: Plenary Conference: “Mechanisms of arsenic transport and detoxification in plants”.

Keynote speaker: Fang-Jie Zhao, Rothamsted Research, Harpenden, UK. COST FA 0905.


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15:10-15:30: Oral presentation: “Translocation and biotransformation of applied selenium in brassica and accumulation of valuable selenium compounds” Seppänen M.M., Kontturi, J., López Heras, I., Madrid Y., Cámara, C., Hartikainen, H. Department of Agricultural Sciences. University of Helsinki, Finland. COST FA 0905.

15:30-15:50: Oral presentation: “Regulation of the plant zinc deficiency response”. Ribeiro Henriques, A., Campos A.C.A.L., Lin, Y.F., Schat, H., Assunçao, A.G.L., Aarts, M.G.M. Laboratory of Genetics, Wageningen University, The Netherlands. COST FA 0905.

15:50-16:20 Coffee break.

16:20-16:40: Oral presentation: “Zn-/Fe-cross homeostasis involved in the generation of the Zn-related phenotype of transgenic tomato”. Barabasz, A., Antosiewicz, D.M. University of Warsaw, Faculty of Biology, Institute of Plant Experimental Biology, Poland. COST FA 0905.

16:40-17:00 Oral presentation: “Lessons from Cd Hyperaccumulators”. Verbruggen N. Laboratory of Plant Physiology and Molecular Genetics, Université Libre de Bruxelles. Belgium COST FA 0905.

17:00-17:15 Closing of Cost Meeting.


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POSTERS

P.1.1. “Effect of phytase addition on iron, cupper and zinc absorption in growing-finishing pig fed diets deficient in total phosphorus”. López C., Madrid, J., Megías, M.D., Martínez, S., Hernández, F. Department of Animal Production, Faculty of Veterinary Science, University of Murcia, Spain.

P.1.2. “Effect of phytase addition in growing pig diets on tissue mineral concentration”. López, C., Madrid, J., Megías, M.D., Valverde, M., López, M., Hernández, F. Department of Animal Production, Faculty of Veterinary Science, University of Murcia, Spain.

P.1.3. “Determination of the coefficients of boron absorption from broiler diets supplemented with boric acid”. Untea, A.E., Criste, R.D., Olteanu, M., Cornescu, G. M., Duca, R.C. National Research and Development Institute for Biology and Animal Nutrition, Balotesti, Romania.

P.2.1. “Effect of consuming zinc-fortified bread on serum zinc and iron status of zinc-deficient women”. Nekouei, N., Fazilati, M., Shahedi, M. Department of Food Science and Technology, Isfahan University of Technology, Isfahan, Iran.

P.3.1. “Use of antioxidants against kidney damage caused by arsenic intoxication during pregnancy and lactation” Pineda, J., Herrera, A., Antonio, M.T. Department of Physiology (Animal Physiology II), Faculty of Biology, Complutense University, Madrid, Spain.

P.3.2. “Hepatic damage produced by low doses of arsenic intoxication during pregnancy and lactation: antioxidants treatment”. Pineda, J., Herrera, A., Antonio, M.T. Department of


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Physiology (Animal Physiology II), Faculty of Biology, Complutense University, Madrid, Spain.

P.3.3. “Analysis of heavy metals from ecological and nonecological alcoholic beverages from North West of Spain”. Ordoñez, C., Cubría, J.C. Department of Biomedical Sciences, University of León, Spain.

P.3.4. “Cadmium daily intake from lettuce plants grown in tropical contaminated soils”. Nogueira, T.A.R, Abreu Junior, A., He Z.L., Alleoni, L.R.F., Trombeta, A.L.B. Malavolta, E. University of Sao Paulo (CENA/USP), Piracicaba, SP, Brazil.

P.3.5. “Arsenic, cadmium, lead and mercury content in foodstuffs of animal origin in Finland in 2010”. Venäläinen Eija-Riitta. Finnish Food Safety Authority, Chemistry and Toxicology Unit, Evira, Helsinki, Finland.

P.3.6. “Ultrastructural and microanalytical study of lactating mammary gland tissue after terbium administration”. Ayadi, A., Maghraoui, S., Abdessalem, S., Ben Hadj, F., Tekaya, L. Laboratoire de Physiologie, Faculté de Médecine de Tunis, Tunisie.

P.3.7. “Intracellular behaviour of indium and samarium in the lactating mammary gland cells”. Maghraoui, S., Ayadi, A., Abdessalem, S., Ben Hadj, F., Ben Ammar, A., Jaaoura, M-H., Tekaya, L. Laboratoire de Physiologie, Faculté de Médecine de Tunis, Tunisie.

P.3.8. “Reducing Cd accumulation in sweet pepper fruits by grafting”. Morikawa, C.K., Saito, T. National Institute of vegetable and Tea Science, National Agriculture and Food Research Organization. Tsu, Japan.


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P.3.9. “Trace element toxicity from an abandoned mining site in a semiarid zone”. García Lorenzo, M.L., Pérez-Sirvent, C., Martínez-Sánchez, M.J., Molina-Ruiz, J. Department of Agricultural Chemistry, Geology and Pedology. University of Murcia. Spain.

P.3.10. “Utilisation of Zn chelate with amino acids in layer diets to reduce Zn excretion in droppings”. Criste, R.D., Untea, A., Cornescu, G.M., Bercaru, A., Iulia, M. National Research and Development Institute for Biology and Animal Nutrition (IBNA-Balotesti) Romania.

P.3.11. “The effect of organic and mineral fertilizers on the contents of metals and polyphenols; case study: grasses from permanent grassland”. Harmanescu, M., Gergen I. Banat´s University of Agricultural Sciences and Veterinary Medicine, Faculty of Agriculture, Romania.

P.3.12. “Determination of total arsenic in marine sediments, waters and fish tissues by atomic fluorescence spectrometry”. Martínez-Sánchez, M.J., González Párraga, P., Cuesta, A., Esteban, M.A., Martínez, S., Pérez-Sirvent, C. Department of Agricultural Chemistry, Geology and Pedology. University of Murcia, Spain.

P.3.13. “Bioaccessibility of trace elements in waste mining materials, SE Spain”. Martínez-Sánchez, M.J., Pérez-Sirvent, C., García-Lorenzo, M.L., González-Ciudad, E., Mantilla, W., Martínez-López, S., Martínez, L.B., Pérez-Espinosa, V. Department of Agricultural Chemistry, Geology and Pedology. University of Murcia, Spain.

P.3.14. “Mechanism of toxicity on human vascular endothelial cells by humic acid obtained from tropical peatlend.” Kaihara, Y., Komori, M., Sun, Y., Sikder, M.T., Hosokawa, T., Saito, T., Kurasaki, M. Graduate School of Environmental Sciences, Sapporo, Japan.


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P.4.1. “Whole amaranth flour: evaluation of nutritive value as breadmaking ingredient.” Sanz-Penella, J.M. and Haros, M. Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Valencia, Spain.

P.4.2. “Effect of alpha-lactalbumin and nucleotides on calcium, iron and zinc uptake and transport in Caco-2 cells from standard solutions”. Peso Echarri, P., Frontela Saseta, C., González Bermúdez, C., García Alonso, J. González, Marín, A., Ros Berruezo, G., Martínez Graciá, C. Department of Food Science and Nutrition, Veterinary Faculty, Murcia, Spain. COST FA 0905

P.4.3. “Influence of different caseinophosphopeptides on ferritin synthesis in Caco-2 cells”. García-Nebot, M.J., Alegría, A., Barberá R., Boujallab, S. Nutrition and Food Chemistry Department. Faculty of Pharmacy, University of Valencia, Spain.

P.4.4. “Investigations on factors impacting iron bioavailability from potato”. Miranda, L., Iammarino, C., Ziebel, J., Evers, D. Centre de Recherche Public-Gabriel Lippmann, Department EVA, Luxembourg. COST FA 0905

P.5.1. “Potassium apigenin: a promising radiation countermeasure”. Alcaraz, M., Castillo, J., Benavente García, O., Armero, D., Alcaraz-Saura, M., Achel, D., Vicente, V., de las Heras, M. Department of Radiology and Physical Medicine, University of Murcia, Spain.

P.5.2. “Carnosic acid: a promising molecula for radiation protection”. Alcaraz, M., Castillo, J., Benavente García, O., Armero, D., Alcaraz-Saura, M., Achel, D., Vicente, V., de las Heras, M. Department of Radiology and Physical Medicine, University of Murcia, Spain.


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P.5.3. “Curcumin and copper binding curcumin induced apoptosis in PC12 cells via mitochodrial pathways”. Nishimura, R., Sato, Y., Tanaka, M., Miyajima, M., Hosokawa, T., Kurasaki, M., Saito, T. Graduate School of Health Sciences, Hokkaido University, Sapporo, Japan.

P.5.4. “Folate levels of vegetables commonly consumed in China”. Shohag, M.H.I., Wey; Y., Yang, X., Zhang, J., Stofella, P.J. MOE Key Laboratory of Environmental Remediation and Ecosystem Health, College of Environmental and Resources Science, Zhejiang University, China.

P.5.5. “Evaluation of selenium and sulphur content in garlic (Allium sativum L.) as a posible source of organocompounds with health benefits.” Ovesná, J., Staveliková H. Hornicková, J., Velisek, J. Crop Research Institute, Prague, Czech Republic.

P.6.1. “Interaction of aluminum and boron in tea pants”. Hajiboland, R., Bastani, S., Bahrami Rad, S. Department of Plant Sciences, University of Tabriz, Iran.

P.6.2. “Effects of selenium suplementation in nodulated alfalfa plants”. Hajiboland, R., Kontturi, J., Rahmat, S., Owusu, A., Aiasgharzad, N. Hartikainen, H. Seppänen M.M. Department of Agricultural Sciences, University of Helsinki, Finland.

P.6.3. “Microelements accumulation in the mulberry trees grown in different climatic conditions”. Lacatusu, A.R., Lacatusu, R., Brailoiu, D., Dumitru, M. National Research and Development Institute for Soil Science, Agrochemistry and Environment Protection, Bucharest, Romania.

P.6.4. “Necessity of biofortification of plants used as fodder or vegetable origin food with selenium, in Romania”. Lacatusu,


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R., Lacatusu, A-R., Stanciu-Burilean, M.M., Lungu, M. National Research and Development Institue for soil Science, Agrochemistry and Environment Protection, Bucharest, Romania.

P.6.5. “Biofortification of cultures using phytochelatins”. Navarro Aviñó, J.P. Universidad Jaume I. Castelló de la Plana. Spain. COST FA 0905.

P.6.6. “First report of low selenium concentrations in soil and bread-making wheat grain in Guadalquivir Valley (Southern Spain)”. López-Bellido, F.J., Poblaciones, M.J., Rodrigo, S., López-Bellido, L., Zhao, F.J. Dpto. Producción Vegetal y Tecnología Agraria. University of Castilla-La Mancha. Spain.

P.7.1. “Trace elements in black, green, red and white tea leaves”. Pérez Marrero, B., Gara, L., Rubio, C., Hernández Sánchez, C, Gutiérrez, A., Hardisson, A. Área de Toxicología de la Universidad de La Laguna, Tenerife, Spain.

P.7.2. “Forensic analysis of human teeth by laser-induced breakdown spectroscopy”. Fortes, F.J., Pérez-Cárceles, M.D., Sibón, A., Luna, A., Laserna, J.J. Department of Analytical Chemistry, Faculty of Sciences, University of Málaga, Spain.

P.7.3. “Cu and Zn in 2009 wines from 5 Canary Islands Designation of Origin” Naoual B.C., Gara, L., Rubio, C., Méndez, J.M., Gutiérrez, A., Hardisson, A. Área de Toxicología de la Universidad de La Laguna, Tenerife, Spain.

P.7.4. “Mineral composition of wild and cultured Sea bass (Dicentrarchus labrax, L.)”. Santaella, M. Martínez Graciá, C., Periago, M.J. Department of Nutrition and Food Science. Faculty of Veterinary Science, University of Murcia, Spain.


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P.7.5. “Use of ultrasound-assisted dispersive liquid-liquid microextraction followed by electrothermal atomic absorption spectrometry for the speciation of chromium released from toys”. López-García, I., Briceño, M., Campillo, N., Hernández-Córdoba, M. Department of Analytical Chemistry, Faculty of Chemistry, University of Murcia. Spain.

P.8.1. “Natural genetic variation controlling zinc use efficiency in Arabidopsis”. Campos, A.C., Henriques, A.R., Moreira, C.N., Assuncao, A.G., Aarts., M. Wageningen University, Laboratory of Genetics, The Netherlands. COST FA 0905.

P.8.2. “Effects of heavy metals on the detoxification system of Typha latifolia”. Schröder, P., Lyubenova, L. Helmholtz Zentrum München German Research Center for Environmental Health, Department Microbe-Plant Interactions, Neuherberg, Germany. COST FA 0905.

P.8.3. “Use of alkaline materials in humic podzols: A way to reduce Cd in vegetables and cereals”. Mench, M., Verdal, M-N., Solda, P., Lecuyer, I. UMR BIOGECO INRA 1202, Ecology of Communities, University of Bordeaux 1, France. COST FA 0905.

P.8.4. “Preventing toxic element penetration into plants and fruits by using grafted plants.” Edelstein, M., Plaut, Z., Ben-Hur, M. Department of Vegetable Crops and Plant Genetics. Agricultural Research Organization. Newe Ya’ar Research Center, Israel. COST FA 0905.

P.8.5. “Understanding basis of magnesium use efficiency in plants”. Herman, C., Verbruggen, N. Lab of Plant Physiology and Molecular Genetics, Université Libre de Bruxelles, Belgium. COST FA 0905.


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P.8.6. “Current level of lead and cadmium in edible and pasture plants growing in areas with elevated metal content and methods for diminishing metal concentration in plants”. Gzyl, J., Pogrzeba, M., Krzyzak, J. Institute for Ecology of Industrial Areas, Katowice, Poland, COST FA 0905.

Session I: Trace elements in livestock

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Trace elements and animal health: what’s new?

Jerry W. SpearsNorth Carolina State University, Raleigh, NC, USA

This presentation will focus on recent advances in iron and copper metabolism of domestic animals. A number of transport proteins have been characterized that are involved in cellular uptake and effux of iron and copper. Divalent metal transporter 1 (DMT1) is the major protein that imports Fe into the small intestine while ferroportin is involved in export of Fe from tissues. Expression of both of these transporters are increased by Fe deficiency but decreased by high dietary Fe. DMT1 also transports manganese (Mn), and competition for DMT1 may explain the antagonism between Fe and Mn. High Fe concentrations in some feedstuffs may be a problem in animals. High dietary Fe (500-1,000 mg/kg diet), provided from ferrous sulfate, reduces Mn and Cu status, and also increases intestinal permeability in young calves and pigs. Ruminants can be exposed to high Fe via soil ingestion or soil contamination of forages. Research suggests that acid conditions occurring during fermentation of silage and haylage greatly increases bioavailability of soil Fe in ruminants. Legumes are often high in Fe and much of the Fe in legumes is present in


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the form of ferritin. Ferritin from soybeans has been shown to be absorbed as well as ferrous sulfate in humans. Transporters involved in the import and export of Cu from the intestine and liver will be discussed as well as Cu chaperone proteins that deliver Cu to specific cellular targets. Genetic differences in Cu metabolism among cattle breeds may relate to differences in expression of certain transporters. Protein and mRNA expression of copper transporter 1, an important protein in cellular uptake of Cu by enterocytes, has been found to be lower in the duodenum of Simmental compared to Angus cattle. The Cu chaperone protein that delivers Cu to Cu/Zn superoxide dismutase in the cytosol is increased by Cu deficiency in cattle.

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Interactions between gut infections and copper-induced hepatotoxicity in ruminants: possible mechanisms and

implicationsN. F. Suttle

Moredun Foundation, Penicuik EH26 9DU, UK

Ruminants have a unique capacity for hepatic Cu storage to counter low Cu availability in forages. However, superior availability of Cu in cereal-based diets can cause hepatotoxicity followed by a haemolytic crisis in sheep. Factors that precipitate the sudden release of stored copper are unknown. Bovine copper toxicity may be under-diagnosed in the absence of a haemolytic stage. New evidence is presented that gut infections influence hepatic copper storage, protecting against or precipitating hepatotoxicity, depending on the timing of infection.

Loss of a 1 y.o. lamb from a haemolytic crisis in an investigation of humoral and cellular immune responses to perinatal Mycobacterium avium infection prompted a biochemical assessment of subclinical copper poisoning in three groups of 5 or 6 cohorts and its responsiveness to antidote (ammonium tetrathiomolybdate,


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ATTM). Raised plasma enzyme activities confirmed widespread hepatopathy, which was less severe and more responsive to ATTM in two infected groups than in controls. Data from a previous study indicated that the infection, which causes chronic intestinal inflammation, more than halved liver Cu, presumably by increasing copper turnover and /or decreasing copper absorption.

Excessive copper feeding in a dairy herd caused calves to be born with high liver Cu. Perinatal mortality was high in the herd and losses were associated with mild evidence of gut infection and mild hepatotoxicity, neither abnormality being considered fatal. Reduction of Cu supplementation was followed by a reduction in mortality and lowering of liver Cu in the newborn. When copper has accumulated prior to infection, the lysosomal store may be de-stabilised and survival compromised.

Chronic infections may precipitate haemolytic crises in copper-loaded sheep and increase requirements for Cu and other elements in otherwise normal circumstances.

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The impact of EU legislation on trace element and mineral supplementation of

livestockAdrian Packington

DSM Nutritional Products Ltd., Heanor Gate, Heanor, Derbyshire, DE75 7SG, UK.

[email protected]

David PickardDP Consulting, B-Hive Business Centre, Saltburn-By-The-Sea, Cleveland,

TS12 2LQ, [email protected]

The trace element and mineral nutrition of livestock has always been influenced by legislation affecting both choice of raw materials and their conditions of use. However, in recent years the scope of legislation has broadened and become more focussed on specific nutrient materials. The rate of change has rapidly accelerated and the impact in all areas has been magnified.

In general there are two broad categories of legislation. Firstly, there is legislation affecting the sale, supply and use of all nutrient materials in all environments; this category includes REACH, Packaging and Labelling, Transport and Supply and Health and Safety regulations. Secondly there is legislation specific to the


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animal nutrition field which restricts nutrient sources to certain substances, requires formal authorisation of all trace elements and their conditions of use, limits the content of undesirable substances and affects the labelling of feed additives, premixtures and feedingstuffs.

For certain mineral feed materials, current heavy metal limits are difficult to achieve. Further reductions in current maximum permitted levels of lead, cadmium, and fluorine are expected and which may limit supply and increase costs. All trace elements are currently the subject of a far-reaching and costly reauthorisation process and the required safety assessments may reveal specific issues. For example, some trace element sources are very toxic and dusty, posing significant worker safety risks, whilst others at currently legal inclusion rates may require expensive ecotoxicity studies or more likely be subject to restricted inclusion rates.

Both categories of legislation outlined affect the entire supply chain and current requirements and future changes will impact on premixture and feed formulation choices and costs. This paper examines some of these issues in more detail.

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Iron and zinc content in meat from cattle with chronic fascioliasis

Sánchez, P.1, Ros, G.2, Martínez Graciá, M.C.2

1Department of Anatomy and Comparative Pathology Anatomy.Veterinary Faculty. University of Murcia. Spain.

2Department of Food Technology, Nutrition and Bromatology. Veterinary Faculty. University of Murcia. Spain.

Background/Objectives: Fascioliasis –a disease of herbivores caused by the trematode Fasciola hepatica in Europe– has been characterised by hepatobiliary lesions being ordinary diagnosed in slaughtered cattle. The most common carcasses of infested animals do not show any alterations being just the liver condemned. The aim of the present study was to investigate if the disorders caused by this parasite in the bovine host could affect the concentration of iron and zinc in muscle by influencing the nutritional quality of beef.

Subjects/Methods: We determined concentrations of iron and zinc by atomic absorption spectroscopy in samples taken from diaphragmatic crura and pectoral muscle of twenty-two bovines slaughtered between eight and fourteen months old in an Alicante’s abattoir (Spain). From the details provided eleven showed macroscopic liver lesions being compatible with chronic


50

fascioliasis during the routine meat inspection. The other eleven healthy bovines, were from the same age, sex, region of birth and feedlot finishing farm than the ones mention above but without fascioliasis.

Results: For the present study significant differences (p<0.05) in iron and zinc concentration were found between meat from diaphragmatic crura from infested and non-infested bovines (iron: 3.75 vs. 4.51 mg/100 g; zinc: 3.48 vs. 3.92 mg/100 g), resulting 16.91% less of iron and 11.17% less of zinc. On the other hand, fascioliasis did not influence significantly in these two minerals analysed in meat samples of pectoral muscle. However, the iron mean concentration was 16.27% lower in parasitized animals (1.71 vs. 2.04 mg/100 g) while zinc concentration was similar in both groups of animals (3.25 vs. 3.18 mg/100 g).

Conclusions: To our knowledge, the contribution of beef to the Recommended Daily Allowance of iron and zinc is significantly lower if the meat belongs to cattle which shows lesions of chronic fascioliasis although this fact depends on the meat cut.

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Excess iodine intake by the ewe in late pregnancy reduces immunoglobulin G

absorption in the newborn lamb Boland, TM.

School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland

The lamb is born hypoimmunocompotent and is dependent on the postnatal absorption of antibodies from colostrum to confer disease resistance in early life. A number of maternal and nutritional factors influence this transfer, but perhaps none so dramatically as high level iodine intake by the pregnant ewe in late gestation. The following findings represent the outcomes of a series of studies conducted over a number of years. High level iodine intake by the pregnant ewe (11 to 40 mg/ewe/day) during the last six weeks of pregnancy greatly reduces the ability of the newborn lamb to absorb colostral immunoglobulin G (IgG). While these levels of intake are greatly in excess of requirements they do represent the voluntary iodine intake of ewes when offered free access mineral blocks. In addition to reduced IgG absorption, vitamin E absorption from colostrum is also reduced. The response to this high level iodine intake is rapid with supplementation during the final two weeks of


52

pregnancy sufficient to produce the response. A two week supplementation period earlier in pregnancy has no effect. This is a dose dependent response with greater reductions seen at higher levels of intake. The lamb is preprogrammed in utero for this effect and the quality or availability of colostral IgG is unaffected by treatment. In the small intestine, the primary site of IgG absorption there are alterations in mRNA expression of a number of genes, namely β2-microglobulin (B2M) and a lower expression of upstream stimulator factor 2 (USF2), interleukin-4 (Il-4) and thyroid hormone receptor β (THRβ) (P < 0.05) potentially mediated through alterations in thyroid hormone metabolism. These findings indicate pathway involved in the observed reduction in IgG absorption and the need for a downward revision (to approximately 40% of current values) of the iodine toxicity levels for ewes during late pregnancy.

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Veterinary and public health implications of cattle farmed in the

vicinity of a vanadium processing plantBotha C.J.1, Gummow B.2

1Faculty of Veterinary Science, University of Pretoria, Onderstepoort, South Africa, 2School of Veterinary and Biomedical Sciences, Faculty of Medicine,

Health and Molecular Sciences, James Cook University, Townsville, Australia

Vanadium is mined in the northern provinces of South Africa. Cattle are farmed in the immediate surroundings of mines or processing plants and are exposed to vanadium to a varying degree. A potential for vanadium to enter the food chain exists when these cattle are slaughtered or if milk is obtained from exposed cows. In this long-term study 42 adult Brahman-cross cattle were assigned to two groups and were farmed extensively directly adjacent to and 2 km from a vanadium processing plant, respectively. Throughout the five year period the cattle were monitored for signs of intoxication. The adult cattle showed no evidence of vanadium poisoning, but the calves became ill and exhibited severe upper respiratory tract infection. This was interpreted that the cattle were exposed to vanadium at close to the no observable adverse effect level. The potential average exposure doses of vanadium (mg/kg/day) were calculated


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for each animal. During this period 6 cohorts of animals were slaughtered at a nearby abattoir. The median exposure doses in the month prior to slaughter ranged from 0.55 – 2.73 mg vanadium/kg/day. Liver, kidney and muscle samples were collected from the cattle at slaughter to determine vanadium concentrations. In addition, vanadium concentration in the milk of cows sampled over the five year period was also determined. The concentrations of vanadium in the edible tissues ranged from <0.05 to 11.51 mg/kg (wet mass basis) and the median concentration of vanadium in the milk was 0.23 mg/L. Concentrations were further modelled using a lognormal distribution function to predict possible extreme values and to calculate the potential daily intake of probable amounts of vanadium by consumers ingesting these foodstuffs. People drinking milk were most at risk. The model predicted that there is less than a 5% chance that the potential daily intake of vanadium from milk will be greater than 44 μg/kg/d for adults. Based on current knowledge of toxicity in humans it was concluded that the tissue and milk residues from cattle should pose no health risk to the consumer.

Session II: Trace elements as food fortificants

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New developments in food fortification Michael B. Zimmermann, M.D.

Human Nutrition Laboratory, Institute of Food, Nutrition and Health, ETH Zurich, Switzerland

In 2011, we have new opportunities to correct micronutrient deficiencies using food fortification. We have new compounds, such as nanostructured iron and zinc compounds with high bioavailability and sensory stability in foods. We have identified new target groups for fortification, such as weaning infants at risk of iodine deficiency, even in countries with established iodized salt programs. But with these new opportunities come new challenges. We know the increasing prevalence of obesity in industrialized and transition countries may impair iron metabolism and absorption, and obese individuals have a higher risk of iron deficiency. Common infections in tropical regions also impair iron absorption and may blunt the effect of fortification programs. And we also have new safety concerns, including the modification of the gut microbiome to a potentially more pathogenic profile by iron fortification. Therefore, although micronutrient programs are extremely cost effective, and the 2004 World Bank Report on correcting micronutrient deficiencies stated “Probably no other technology today offers as large an opportunity to improve lives and accelerate development at such


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low cost and in such a short time”, we still need to answer many scientific questions in order to ensure safe and effective program implementation.

59

Application of nanotechnology in Agriculture and Health: Progress and

perspectivesHe Z. L.,1 Yang X. E.,1,2 Bakshi S., and Stoffella P. J.1

1 Indian River Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, Fort Pierce, FL 34945-3138 zhe@

ufl.edu; 2 MOE Key Lab of Environmental Remediation and Ecosystem Health, College of Environmental & Resources Science, Zhejiang University,

Hangzhou 310029, China

Abstract

Nanotechnology deals with structures sized between 1 to 100 nm in at least one dimension. It involves developing materials or devices within that size. Since its emergence in the beginning of the 21 century, nanotechnology has had wide applications in agricultural production, food preservation, environmental quality, and human health. Organic and inorganic nanoparticles of natural origin are present in abiotic and biotic systems and play an important role in storing nutrients in soil, removing contaminants from water, and catalyzing or directly participating in many chemical and biochemical processes in plant, animal, and humans. In recent years, different kinds of nanoparticles have been artificially created and applied for improving crop


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yield and quality, remediating environment, enhancing food production, and delivering drugs/nutrients to target sites in humans. For instance, nano green, an oil based nano micelle was observed to increase rice yield by 10-20% and improve fruit quality of many horticultural crops; zero value iron nanoparticles have been used to remove arsenic in groundwater; carbon nanotubes are capable of removing organic pollutants from waters; and silver nanoparticles are increasingly applied in cosmetic products and paints because of their being antimicrobial and sanitary. Currently, great interests are being attracted to the development of smart nanoparticle systems which can deliver drugs, pesticides, and nutrients including trace elements directly to the target sites in plants and humans so that effective compounds can be applied with needed concentrations without causing toxicity. These systems will have great potential for trace element application considering that current utilization efficiency of the trace elements is very low. However, there is also increasing concern regarding the effects of some manufactured nanoparticles on environmental quality and human health. Regulations governing the use and disposal of nanomaterials are lacking. More research and regulatory efforts are required to ensure the beneficial application of nanotechnology.

Session III: Toxicology and risk assessment of trace elements

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Movement of heavy metals from soil to edible parts of crops (Human food

Chain) and risk AssessmentXiaoe Yang1,2, Zhenli He2, Wendan Xiao1, Mahamad Tarig1

1MOE Key Laboratory of Environmental Remediation and Ecological Health, College of Environmental and Resource Sciences, Zhejiang University,

China; 2Indian River Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, Fort Pierce, FL34951-3138, USA.

[email protected]

Heavy metals, such as cadmium, copper, lead, chromium and mercury, are important environmental pollutants, particularly in areas with high anthropogenic pressure. Their presence in the atmosphere, soil and water, even in trace amounts can cause serious problems to all organisms, and heavy metal bioaccumulation in the food chain can be highly dangerous to human health. Heavy metals enter human body mainly through two routes namely: inhalation and ingestion, ingestion being the main route of exposure to those elements in human population. Heavy metal intake by humans through food chain has been reported in many countries. For soil-plant-food chain system, heavy metal toxicity risk assessment is based on soil threshold that does not exceed food hygiene standards, and is highly dependent on the


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movement of heavy metal from soils to the edible parts of the crops. The movement of heavy metals from soil to edible parts of crop plants is dependent on metal kind, soil type, crop species or cultivars, and microbial interactions. Cadmium is among most mobile in food chain of all heavy metals. Soil properties such soil pH, organic matter content, and clay mineral are the major factors regulating metal bioavailbility and accumullation in food chain. In addition, the interactions of soil-plant root-microbes play important roles in regulating heavy metal movement from soil to the edible parts of crops. Some endo-microbes has been found to increase metal movement and accumulation into crop by several times. Agronomic practices such as fertilization, water management, and crop rotation influence bioavailability and crop accumulation of heavy metals, thus affecting the thresholds of assessing dietary toxicity of heavy metals in the food chain. This paper reviews the movement of heavy metals from soil to human food chain in vegetables and food crops, and risk assessment for potential dietary toxicity.

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Toxicology and trace metals risk Assessment: a total diet study of the

Spanish populationCarmen Rubio Armendáriz, PhD

Toxicology DepartmentLa Laguna’s University

S/C de Tenerife, Islas Canarias, España

Food toxicology is the study of the nature, properties, effects, and detection of toxic substances in food, and their disease manifestation in humans. Risk analysis is broadly defined to include risk assessment (scientific evaluation of the probability of harm resulting from exposure to toxic substances), risk characterization (a description of the nature and magnitude of health risk that combines results of exposure assessment and hazard identification and describes the uncertainties associated with each step), risk communication, risk management, and risk control policy.

Trace metals (Fe, Mg, Li, Zn, Cu, Cr, Ni, Co, Va, As, Mo, Se) are necessary for good nutrition. Diet is the major intake source of trace elements by human beings. Minerals can be naturally present in foods or added to them both for technological or nutritional


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purposes (fortified foodstuffs). The dietary intake levels of these metals have caused concern both for being potentially too low, and also because of the adverse effects that excessive intakes of certain minerals may cause.

The Population Reference Intakes (PRIs), also called Recommended Daily Allowances (RDAs), are based on the principle that most, if not all, individuals of a population or a specific population group should consume adequate amounts of each nutrient to satisfy their requirements. Such recommended values are generally based on the principle of the average requirement plus two Standard Deviations (SD) for the nutrient in the population group.

Zn is and essential trace element in human metabolism and it therefore has a recommended daily allowance (RDA). Despite evidence that they are also essential, Cu and Mn have no established RDA, although they do have estimated safe dietary daily intake limits. Occasional studies have asserted that Ni is also nutritionally essential but it currently has no established RDA.

The European Union has established a Population Reference Intake (PRI) of 1.1 mg Cu for adults. The European Commission Scientific Committee on Food (SCF) has issued an opinion on Tolerable Upper Intake Levels of Cu and derived a UL of 5 mg/day for adults and 1 - 4 g/day for children. While the European Population Reference Intake (PRI) for zinc, for adult males and females, is 9.5 mg/day and 7.0 mg/day, respectively, the RDA is 15 mg Zn/d in the United States. The SCF has established a UL for zinc, from all sources, of 25 mg/day. For Mn, 1 - 10 mg/day is considered as an acceptable range of intake, and a supplemental intake of up to 4 mg Mn/day (equivalent to 0.07 mg/kg bw for a


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60 kg adult), in addition to the diet, would be unlikely to produce adverse effects in the general population.

There is no doubt that it is increasingly difficult to develop accurate assessments of the pattern of the overall diet of a given population, since these vary by region, between groups within each population, and over time. Furthermore, the risk assessment of trace metals intake levels involves the comparison of the RDA with the estimated intake of the population, and these estimated intake studies are based on dietary survey data and the relevant food composition data, both of which need to be updated constantly.

In the Canary Islands, the daily dietary intakes of four essential metals (Cu, Zn, Mn and Ni) were estimated. Food groups and food consumption data were obtained from the last official nutritional survey in the Canary Islands. Trace metals contents were determined in 420 food and drink samples collected in local markets. Inductively Coupled Plasma - Atomic Emission Spectrometry (ICP-AES) was used for the analysis of Cu, Zn and Mn, and Atomic Absorption Spectrometry with graphite furnace (GF - AAS) was used to determine Ni in foods and drinks.

The estimated daily dietary intakes of Cu, Zn, Mn and Ni in the Canary Islands were 2.098 mg/day, 8.954 mg/day, 2.372 mg/day, and 0.0925 mg/day respectively. Mean dietary Cu intakes in different European countries have been estimated in the range of 1 - 2.2 mg/day on average, and as high as 1.2 - 4.2 mg/day. Mean intakes of Zn in adults in Europe have been found to range from 7.5 - 12.1 mg/day. According to total diet studies the average and 97.5th percentile Mn intakes from food in European countries is in the range of 2.3 to 4.9 mg/day and 4.8 to 8.2 mg/day, respectively.


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While Cu and Mn dietary intakes in the Canary Islands met the RDAs, the mean daily intake of Zn was below the RDA. Cereals were found to be the food group that contributed most to the intake of these metals.

Recommended levels of metal intake can only be established after determining and monitoring the metal contents in foods and drinks. Very few studies have been conducted about overall dietary intake of trace metals. Therefore further studies should be conducted to help inform the risk communication and risk management decision-making processes related to trace metals hazards in food.

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Presence and distribution of trace elements and heavy metals in adult

antarctic penguins: background levels?Jerez S.1, Motas M.1, Palacios, M.J.2, Cuervo J.J.3, Díaz J.4,

Benzal J.3, Vidal V.3, Barbosa A.2

1 Toxicology Area, Faculty of Veterinary Medicine, University of Murcia, Spain.2 National Museum of Natural Sciences, Spanish Council for Scientific

Research, Madrid, Spain.3 Estación Experimental de Zonas Áridas, CSIC, Almería, Spain.

4 Cepave-Conicet- National University La Plata, La Plata, Argentine

Antarctica is often considered as one of the last pristine regions in the world, and the concentrations of trace elements detected in its ecosystems have been taken as background levels to evaluate pollution in other areas. However, the Antarctic environment can be affected by persistent pollutants at global scale since they can be transported until remote areas through oceanic and atmospheric ways. In addition, human contaminant activities have been increased at local scale during the last years, especially in the Antarctic Peninsula.

To study the current levels of trace elements and heavy metals in the Antarctic Peninsula, we used samples of three species of penguins: Pygoscelis papua, antarctica and adeliae. As they are long-


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lived, have a permanent ecological niche and dominate among the antarctic bird biomass, penguins have been pointed as potential biological indicators for monitoring the contamination in Antarctica.

Samples of liver, kidney, muscle, bone, feather and stomach content (n = 64) were collected in three different islands along the west cost of the Antarctic Peninsula (range 62º15’S - 67º46´S, summer seasons 2007-2010). Elements levels were determined by ICP-MS and data were analyzed by SPSS 15.0.

Elevated cadmium levels have been detected in soft tissues of adult penguins from Deception and Avian Islands, especially in kidney (263.93 ± 139.77 and 351.84 ± 0.08 μg/g dry weight, respectively), exceeding normal and even toxic thresholds described for birds. These cadmium levels (3-300 times higher than those detected in stomach contents) seem to be the result of a strong bioaccumulation phenomenon. High cadmium concentrations were accompanied by increases of selenium and zinc levels (Spearman rho = 0.797 and 0.727, p < 0.01), which could be related to a protective mechanism. Other elements were mainly accumulated in liver (Fe, Cu), bone (Ni) or feather (Pb), although they showed a lower organotropism than cadmium.

Supported by: Spanish Ministry of Science and Innovation and European Regional Development Fund (grants CGL2004-01348, POL2006-05175 and CGL2007-60369). S.J. was supported by a grant from Caja de Ahorros del Mediterráneo.

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Renal effects of low environmental exposure to cadmium: evidence of

reverse causation Alfred Bernard and Agnès Chaumont

Unit of Toxicology and Applied Pharmacology, Catholic University of Louvain, Avenue E. Mounier 53, bte 5302, B-1200 Brussels, Belgium. E-mail:

[email protected]

Objectives

Associations of low-molecular-weight (LMW) proteins in urine with low-level urinary cadmium (CdU) are currently interpreted as the sign of renal dysfunction induced by Cd. Few studies have considered the possibility that these associations might arise by a reverse causation in which factors unrelated to Cd would affect the kidney function and thereby the renal elimination of the metal.

Methods

We examined several groups of children (n=284), adolescents (n=738) and adults (n=190) with low environmental exposure to Cd as reflected by CdU levels (<1 μg/g creatinine). We measured the urinary concentrations of Cd, retinol-binding protein (RBP)


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and albumin. Associations between urinary proteins and CdU were assessed by simple and multiple regression analyses.

Results

The median concentrations of CdU (μg/g creatinine, range) in children, adolescents and adults were 0.11 (0.020-0.53), 0.37 (0.087-0.99) and 0.49 (0.07-0.98), respectively. In all studied groups, the concentration of RBP in urine was consistently associated with CdU through linear relationships, which did not vary with gender and age. Further analyses showed that associations between RBP and Cd in urine varied with the integrity of the glomerular function as they were abolished in subjects with increased urinary albumin (competitive inhibition). The albumin excretion also correlated with CdU but only among subjects with low urinary RBP.

Conclusions

A reverse causality due to the co-excretion of metallothionein (the main Cd binding protein) with other low molecular proteins appears as the most logical explanation for these findings. This also suggests that at low environmental exposures, CdU would be more a reflection of the tubular reabsorption capacity of LMW proteins than of the past or current exposure to Cd.

Supported by the European Union (Phime project).

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Cadmium daily intake from lettuce plants grown in tropical

contaminated soilsNogueira T.A.R.1, Abreu Junior C.H.1, He Z.L.2, Alleoni L.R.F.3,

Trombeta A.L.B.3, Malavolta E.1†

1University of Sao Paulo (CENA/USP), Piracicaba, SP, Brazil. Email [email protected], [email protected]. †In memoriam.

2Indian River Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, 2199 S. Rock Road, Fort Pierce,

FL 34945. Email [email protected] of Sao Paulo (ESALQ/USP), Piracicaba, SP, Brazil. Email

[email protected], [email protected]

Cadmium (Cd) is one of the most well-known environmental intoxicants to humans. Estimation of daily Cd intake is important for monitoring environmental Cd pollution and effects on human health. The objective of this study was to evaluate Cd availability for lettuce plants grown in tropical soils contaminated with Cd, using the estimation of Cd daily intake. The experiment was carried out under greenhouse conditions in Piracicaba, state of São Paulo, Brazil. Lettuce plants (two plants/pot) were cultivated in pots filled with 3 dm3 of two Oxisol (Typic Hapludox and Rhodic Hapludox) samples using a randomized block design in a factorial scheme (5 x 2) with three


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replicates. Treatments consisted of Cd rates: 0.0, 0.5, 1.3, 3.0, and 6.0 mg dm-3 (as CdCl2), based on the guideline established by the Environmental Agency of the State of Sao Paulo, Brazil - Cetesb. Daily Cd intake from lettuce was calculated by Cd content in lettuce leaves multiplied with daily lettuce consumption (25; 50; 100; 150; 200 and 250 g, fresh weight). To evaluate the health risk of this estimated dietary exposure, it was compared with the provisional tolerable weekly intake (PTWI) recommended by the Joint FAO/WHO Expert Committee for Food Additives (JECFA). Based on the alert value for Cd in the soil (1.3 mg kg-1) and on the daily lettuce consumption, it was estimated that the daily intake of Cd ranged from 16.2 to 354.7 μg person-1 day-1. For a person with a body weight of 70 kg, the dietary intake of Cd is lower than the PTWI when the daily lettuce consumption were 150 g for Rhodic Hapludox and 50 g for Typic Hapludox. These two soils had high availability to the lettuce plants, and this information deserves attention when managing Cd-polluted soils.

Keywords: dietary intake, human health, soil pollution, trace element

Supported by: this study was, in part, supported by a Ph.D graduate scholarship awarded to the first author (process # 07/59402-0) from FAPESP (The State of São Paulo Research Foundation) and by the University of Sao Paulo and University of Florida.

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Relation of fish consumption to mercury, selenium and arsenic levels in cord

blood and breast milk from Slovenian pregnant women

Ana Miklavčič1, Darja Mazej1, Janja Tratnik1, Alfred B. Kobal1, Mladen Krsnik2, Petra Planin ek1,

Jo ko Osredkar2, Milena Horvat1

1 Jozef Stefan Institute, Department of Environmental Sciences, Jamova39, 1000 Ljubljana, Slovenia

2University Medical Centre Ljubljana, Institute of Clinical Chemistry and Biochemistry, Zaloška cesta 2, 1000 Ljubljana, Slovenia

Fish can represent an important source of mercury, selenium and arsenic in humans. In order to assess possible intake of these elements through fish consumption and their interactions in cord blood and milk, a study involving 447 Slovenian women was implemented in the framework of the the EU 6th Framework programme Public health impact of long-term low-level mixed element exposure in susceptible population strata (PHIME).

Cord blood and breast milk samples were collected from 446 and 287 women respectively. Total mercury (THg) in cord blood was determined by thermal combustion, amalgamation and atomic


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absorption spectrometry using the Direct Mercury Analyser (DMA-80). THg in breast milk was determined using cold-vapour atomic absorption spectrometer (CVAAS). Arsenic in breast milk and cord blood was determined by inductively coupled plasma mass spectroscopy (ICP-MS). Frequency of fish consumption was assessed by food frequency questionnaires filled out by participating women. At low levels of mercury and arsenic exposure, a significant linear correlations were found between mercury levels and selenium levels (r=0.27, p<0.0001), arsenic and selenium levels (r=0.21, p<0.0001) and arsenic and mercury levels (r=0.36, p<0.0001) in cord blood or between mercury levels and selenium levels (r=0.36, p<0.0001) and mercury and arsenic levels (r=0.28, p=0.0013) in breast milk. In addition, a significant relation was found between frequency of fish consumption and mercury levels in cord blood (rs=0.392, p<0.0001) and between frequency of fish consumption and arsenic levels in cord blood (rs=0.24, p<0.0001) or arsenic levels in breast milk (rs=0.23, p<0.0001), possibly explaining the correlations between determined elements in cord blood or breast milk. Furthermore, the found correlations might be due to the protective role of selenium against mercury and arsenic toxicity.

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Evaluation of the assimilation of heavy metals by plants in contaminated soils

submitted to a remediation process1Martínez-Sánchez, M.J., 1Pérez-Sirvent, C., 2Cánovas J.,

1Agudo, I. and 1Banegas, A.1Department of Agricultural Chemistry, Geology and Pedology. University

of Murcia. Spain.2IMIDA – CIFEA. CARM. Murcia Spain.

Email: [email protected]

A greenhouse trial was carried out to evaluate the assimilation of heavy metals by three types of horticultural plants (lettuce, onion and melon), different parts of which are destined for human and farm animals consumption (leaves, roots, fruits).

The plants were cultivated in for types of soil, one of then was contaminated by heavy metals (T1), the second was a uncontaminated soil (blank soil) (T2), the third was the material obtained by mixing T1 with residues coming from demolition and construction activities (T3); while the fourth was the result of remediating T1 with lime residues coming from quarries (T4).

The total metal content (As, Pb, Cd and Zn) of the soils samples, rizosphere, leached water and vegetable samples, were measured


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and the translocation factor (TF), which is defined as the ratio of metal concentration in the leaves to the roots, and Bioconcentration factor (BCF), which is defined as the ratio of metal concentration in the roots to that in soil were calculated.

The statistical analysis of the results obtained showed that these were no significant differences in the heavy metal levels for the vegetables cultivated in T2,T3 and T4. The results for soil sample T1 did not have a normal distribution since the growing of vegetal was not homogeneous and also strongly dependent on the type of cultivation.

Session IV: Bioavailability of trace elements

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Bioavailability of iron from whole diets in adults

Susan J. Fairweather-Tait, Linda J. Harvey, Rachel Collings, Amelie Casgrain, Rachel Hurst and Lee Hooper

Norwich Medical School, University of East Anglia, Norwich, Norfolk, NR4 7TJ, UK

The EURopean micronutrient RECommendations Aligned (EURRECA) Network of Excellence (www.eurreca.org) is generating outputs related to micronutrient requirements. Disparity exists between European countries for many Dietary Reference Values (DRVs) and this may be due to differences in the selection and interpretation of the relevant data for deriving requirements by expert bodies. For some micronutrients where factorial estimates are used to derive the average requirement, for example iron, a ‘bioavailability factor’ must be applied to convert physiological requirements into dietary intake by accounting for variations in absorption and utilisation from the diet. Such factors are often based on absorption estimations from ‘typical diets’.

A systematic review of iron absorption (a surrogate marker of bioavailability) has been undertaken. The review was limited to whole diet studies as single meal studies reportedly exaggerate


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the effects of modulators of iron absorption. A systematic search of the literature and a standardised methodology were employed to identify all potentially relevant studies, and strict inclusion criteria were applied. Due to the influence of iron status on absorption, only studies reporting baseline iron status of the population were included. Assessments of study quality and risk of bias were performed and a minimum 10% duplication of each methodological step was conducted.

A combined search was run with the other EURRECA priority minerals, zinc and selenium, and returned a total of 3211 potentially relevant papers for the three minerals across all population groups. Following an initial screening of titles and abstracts, 440 papers were identified that appeared to measure iron absorption in adults. Of these, following full text assessment, 120 met the inclusion criteria, but only 22 reported absorption from whole diets. These studies were included in the final meta-analysis, and preliminary results will be presented.

Funding: This work was completed on behalf of the EURRECA Consortium and funded under the EU 6th Framework Food Quality and Safety programme, project number FP6-036196-2.

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Selenium uptake, accessibility and bioavailability in wheat

Espen Govasmark, Anicke Brant-Kjelsen, Brit SalbuNorwegian University of Life Sciences – Department of Plant and

Environmental Science, PO-5003, N-1432 Ås, Norway

Food levels of selenium (Se) in Northern Europe are low and agronomic Se-fortification is one strategy to increase the human Se intake. Estimation of diets for Se adequacy requires information on the total Se, but also on Se-species, accessibility, bioavailability of Se-species in food. The work focuses on the distribution and speciation of Se in wheat grains and its accessibility estimated from extraction with human gastro intestinal juices (HGIJ) and Se-bioavailability to chicken determined as the bioconcentration factor (BCF); breast muscle/feed intake Se concentration.

Wheat was Se-selenate enriched by soil or foliar fertilisation at seeding or tillering with or without a spilt nitrogen fertilisation strategy to investigate the Se-species distribution. Field grown Se-enriched wheat were used to estimate the total and Se-species distribution in flour and bran and its accessibility and used chicken feed to estimate the Se BCF and as ingredience in bread to investigate Se-transformation and Se-accessibility from


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wheat flour to bakery product. Se-speciation was performed by anion exchange-HPLC-ICP/MS with 25-250 mM NH4Ac, pH 5.1 gradient elution.

Selenomethionine was the dominant Se-species in grains. The field grown wheat grains contained 1.2 mg Se kg-1, while the flour and bran concentration were 1.1 and 1.6 mg Se kg-1, respectively. Flour-Se was more accessible than bran-Se based on HGIJ extractions. The chicken breast muscle Se-BCF increased from 0.6 to 1.9 by changing from selenite to Se-enriched wheat as dietary Se source. Bread making did not influence the Se-species distribution. The kernel Se-accessibility was higher than in the crust. High bran content in the bread decreased the Se-accessibility. Agronomic Se-fortification to wheat is a promising strategy to increase the human dietary intake.

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Effect of Bifidobacterium phytases on in vitro mineral availability

from infant cerealsSanz Penella1,2, J.M., Frontela2, C., Ros2, G., Martínez2, C.,

Monedero1, V. and Haros1, M.*1Cereal Group and Lactic Acid Bacteria and Probiotics Laboratory, Institute of Agrochemistry and Food Technology (IATA-CSIC), Av. Agustín Escardino 7, Parque Científico, 46980 Paterna-Valencia, Spain; 2Department of Food

Science and Nutrition, Faculty of Veterinary Science and Food Science and Technology, University of Murcia, 30071 Murcia, Spain.

*E-mail: [email protected]

Cereals, incorporated as flours, are usually the first solid foods given to infants between the ages of 4 and 6 months to supplement breast milk and follow-on formula. Mineral bioavailability from infant cereals is usually low owing to the presence of phytate (myo-inositol hexakisphosphate or InsP6). The use of microbial phytases, able to dephosphorylate phytate and usually employed in animal feed, can be an attractive alternative to reduce phytate contents in foods. Phytase activity was recently described in probiotic bifidobacterial strains, opening the possibilities to the use of the corresponding enzymes in foods, due to their QPS/GRAS status. Two commercially available infant cereals (multicereal and gluten-free) were examined by measuring the


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myo-inositol phosphates content and the in vitro Ca, Fe and Zn availability before and after a dephytinization process with purified phytases from Bifidobacterium pseudocatenulatum ATCC 27919 and Bifidobacterium longum spp infantis ATCC 15697. Treatment of the samples with the bifidobacterial phytases reduced the InsP6 contents similar to the treatment with a fungal phytase. The mineral bioaccessibility, measured as the soluble or dialyzable mineral fraction resulting from in vitro gastrointestinal digestion of the samples was estimated. Dephytinization by phytase activities generally increased the in vitro availability of Ca, Fe and Zn for both infant cereals analyzed. The results of this study indicate that dephytinization of infant cereals due to the addition of bifidobacterial phytases could improve the bioaccessibility of minerals.

Supported by Consolider Fun-C-Food CSD2007-00063 Project from Ministry of Sciences and Innovation (MICINN), Spain

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Effect of processing and food matrix on calcium and phosphorus bioavailability

from milk-based fruit beverages in Caco-2 Cells

Cilla A.1, Lagarda M.J.1, Alegría A.1, Sánchez-Moreno C.2, Plaza L.3, Cano M.P.3, De Ancos B.2, Barberá R.1

1Department of Nutrition and Food Chemistry. Faculty of Pharmacy. University of Valencia. Burjassot (Valencia). Spain. 2Department of Food

Characterization, Quality and Safety, Institute of Food Science, Technology and Nutrition (ICTAN-CSIC), Madrid, Spain. 3Department of Food

Biotechnology and Microbiology, Institute of Food Science Research (CIAL, CSIC-UAM), Madrid, Spain.

Objectives: To study if applying emerging technologies, high pressure (HP), as an alternative to traditional thermal treatment (TT) (mild pasteurization) would increase the bioavailability of food components, making the final product more nutritious. Thus, the aim of this study was to determine Ca and P bioavailability from different milk-based fruit beverages as affected by pasteurization and high pressure processing, by means the paired in vitro gastrointestinal digestion /Caco-2 cell model.


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Methods: Nine beverages were studied, all of them containing a mixture of fruit juices (orange, pineapple, kiwi and mango), citric acid and sugar, and differing in the source of milk (whole milk, skimmed milk and soya milk) and the treatment received (non treated, HP treated (400 MPa/ 40 ºC /5 min) and TT (90 ºC/30 s). Samples were subjected to an in vitro gastrointestinal digestion (solubility method) and the bioaccessible fractions used to determine uptake and transport of minerals in Caco-2 cells. Ca was determined by flame atomic absorption spectrophotometry whereas P by visible spectrophotometry. Two-way ANOVA followed by Tukey post-hoc test was applied to the results with a p<0.05 of significance.

Results: Ca bioaccessibility was higher in HP (98.4% ± 1.6%) vs. TT (91.3% ± 1.9%), but Ca bioavailability was equal in all three different matrixes independently of the processing treatment applied. HP treated samples improved P bioaccessibility (98.7% ± 2.5% vs. 87.3% ± 2.2%) and P bioavailability by Caco-2 cells vs. TT samples, being soya milk- and whole milk- based beverages the samples with highest bioavailability values (56.8% ± 1.3% and 40.1% ± 9.9% vs. 15.0% ± 2.1% and 16.8% ± 2.8%, respectively).

Conclusion: HP processing positively modulates Ca and P bioaccessibility and P bioavailability in Caco-2 cells and can be used as an alternative to the traditional heat process in the manufacture of functional foods with improved nutritional value and health benefits.

Supported by: This study has been partially funded by the CONSOLIDER INGENIO 2010 Programme, FUN-C-FOOD CSD2007-063 and AGL2006-12758-C02-01 project.

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Uptake and transport of calcium, iron, and zinc from infant formulas

supplemented with alpha-lactalbumin and nucleotides using Caco-2 Cells

Peso Echarri P., Frontela Saseta C., González Bermúdez C., González Marín A., Ros Berruezo G., Martínez Graciá C.

Department of Food Science and Nutrition, Faculty of Veterinary Science, University of Murcia, Campus de Espinardo 30071, Spain

Breast milk is considered the best food for the newborn during the first months of life, however, when breastfeeding is not possible, infants depend either partially or totally on infant formulas to satisfy their nutritional requirements during their first year of life. Insufficient mineral intake during infancy is responsible for many diseases which can not only influence immediate health, but may also have an adverse impact on adult health. Alpha-lactalbumin and nucleotides are compounds naturally present in breast milk, and nowadays are added to infant formulas as ingredients since have been reported to be important for growth, repair and differentiation of gastrointestinal tract in infants. However, there are little studies on its role on mineral absorption.


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Therefore, the aim of our study was to evaluate the in vitro calcium, iron and zinc availability from two infant formulas (one standard and the other supplemented with alpha-lactalbumin and nucleotides) by the intestinal cell line Caco-2 after an in vitro digestion process. The mineral availability of human milk was studied in parallel because it is the gold standard and a reference for infant formulas. Although the human milk showed a lower mineral content than both infant formulas, the results obtained showed a higher cell transport and uptake efficiency, being indicators of availability. On the other hand the supplemented formula got higher levels of calcium and zinc uptake efficiency than the standard formula (Ca: 3,59% ± 0,41 vs. 2,37% ± 0,41; Zn: 12,23% ± 0,80 vs. 8,605% ± 1,54). We conclude that although mineral availability of human milk is the highest, the addition of alpha-lactalbumin and nucleotides enhances Ca and Zn availability of infant formulas evaluated by Caco-2 cultures.

This work was funded by the Spanish Plan Nacional de I+D+i through projects Consolider Ingenio 2010 Programme (ref. FUN-C-FOOD CSD2007-0623) and AGL-2007-63504.Thanks also to Fundación Seneca for the fellowship of P. Peso-Echarri (07877/BPS/07)

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Iron, calcium and zinc in vitro solubility and availability from different

commercial infant formulas with thickening agents

González Bermúdez C.A.1, Frontela Saseta C.1, Peso Echarri P1., López Nicolás R1., Ros Berruezo G1., Bernal Cava Mª José2,

Martínez Graciá C.1

1Department of Food Science and Nutrition, Faculty of Veterinary Science, University of Murcia, Campus de Espinardo 30071, Spain.

2Institute of Infant Nutrition Hero Baby, Alcantarilla 30820, Murcia, Spain.

Due to lack of maturity of newborn’s digestive system, gastroesophageal reflux (GER) is a common process described in a high rate of infants under 12 months of age. This process is considered as a functional or physiologic one in a healthy infant with no underlying systemic abnormalities. Therefore, infants usually do not require dietary or other therapeutic interventions. Nevertheless, if they are repeatedly described, they may result in substantial nutrient losses because of emesis, being a significant contributing factor to malnutrition. The management of repeated GER episodes includes nutritional modification, such as infant formulas thickened with starches or non digestible carbohydrates. It has been reported that thickening agents, including carob bean


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gum, pregelatinized corn starch or rice starch, may result in a reduction of intestinal absorption of carbohydrates, fats, calcium, iron, zinc and copper. The reason seems to be that cereal contains high amounts of phytates and dietary fibers that depress nutrient bioavailability.

The aim of our study was to evaluate the in vitro calcium, iron and zinc solubility and availability from several commercial infant and follow-on formulas with thickening agent using an in vitro digestion and dialysis process. The results were compared to those obtained with the respective formulas without thickening agent.

The effect of those ingredients on Ca, Fe and Zn solubility and dialysability was different depending on the thickening agent and the type of formula. The most striking effect was found in Calcium dialysability from control follow-on formulas where it was higher than in those with thickening agents (ranging from 9.79% ± 0.33% to 15.96% ± 0.04% and 4.06% ± 0.82% to 4.85% ± 0.11% respectively). Regarding to iron and zinc, the higher lowering effect was showed in soluble fraction whereas dialysability seemed not to be modified. These preliminary results will guide us to study the effect of each of those thickened agents on mineral availability using a Caco-2 model.

This study was funded by Fundación Séneca, Agencia Regional de Ciencia y Tecnología, Región de Murcia (Project 11978/PI/09), and Fellowship of González-Bermúdez (15001/BPS/10).

Session V: Trace elements and health

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Dose-Response meta-analysis to elucidate the relationship between

plasma selenium and prostate cancer riskRachel Hurst1, Lee Hooper1, Teresa Norat2, Rosa Lau2,

Dagfinn Aune2, Darren Greenwood3, Rachel Collings1, Susan J. Fairweather-Tait1

1Norwich Medical School, University of East Anglia, Norwich, Norfolk, NR4 7TJ, UK

2Department of Epidemiology and Biostatistics, School of Public Health, Imperial College London, St. Mary’s Campus, Norfolk Place, Paddington,

London W2 1PG, UK3Centre for Epidemiology and Biostatistics, University of Leeds, Leeds, LS2

9LN, UK

The evidence regarding the relationship between selenium and prostate cancer risk indicates dose- and form–specific effects of selenium, and a potential narrow range of optimal selenium intake and status. The objective of this study was to systematically review and assess the relationship between selenium status and prostate cancer risk, in particular to investigate the dose-response relationship to determine the ‘optimal range’. Data from a meta-analysis of studies included in the World Cancer Research Fund/American Institute for Cancer Research Continuous Update Project database updated to Sept. 2010 will be presented. Meta-


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analyses were performed and study validity, heterogeneity and publication bias were assessed. Dose-response meta-analyses were used, with fractional polynomials for nonlinear trends, to investigate the association between selenium status and prostate cancer risk. Criteria for inclusion included an adult population, assessment of selenium intake or selenium status (including plasma/serum selenium) as an exposure with >2 categories, assessment of prostate cancer cases (number of events) and relative risk as an outcome. There were 8 studies considered for inclusion in the preliminary analysis with >11,000 participants and >4000 incident cases of prostate cancer. The relationship between plasma/serum selenium and prostate cancer was non-linear and risk decreased with increasing plasma/serum selenium up to ~175ng/ml. Study validity and non linear fractional polynomial plots displaying the relationship between plasma/serum selenium and total or advanced prostate cancer risk will be presented.

Funding from the Commission of the European Communities, Research, Technology and Development (RTD) Programme “Quality of Life and Management of Living Resources”, within the 6th Framework Programme (Contract No. FP6-036196-2 EURRECA: EURopean micronutrient RECommendations Aligned Network of Excellence, www.eurreca.org). This report does not necessarily reflect the Commission’s views or its future policy in this area.

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Iron and neurodevelopment and immunity in infants; children and adolescents. A systematic review

Hermoso M.1, Vucic V.2, Vollhardt C.1, Arsic A.2, Roman-Viñas B.3, Iris Iglesia-Altaba4, Gurinovic M.2, Koletzko B.1

1 Division of Metabolic and Nutritional Medicine. Dr. von Hauner Children´s Hospital, Ludwig-Maximilians-University of Munich Medical Centre,

Munich, Germany,2 Centre of Research Excellence in Nutrition and Metabolism. Institute for

Medical Research. University of Belgrade, Belgrade, Serbia,3 Community Nutrition Research Centre of the Nutrition Research

Foundation, Barcelona Science Park, University of Barcelona, Barcelona, Spain,4 Growth, Exercise, Nutrition and Development (GENUD) Research Group,

Escuela Universitaria de Ciencias de la Salud, Universidad de Zaragoza, Zaragoza, Spain,

A systematic review was conducted to summarize the evidence currently available from randomised controlled trials (RCT) concerning the effect of iron intake of infants, children and adolescents until 18 years of age on measures of neurodevelopment, including cognition and psychomotor development, and on immunity. The Cochrane Library, MEDLINE and EMBASE were searched up to and including February 2010. Studies were also identified by checking the bibliographies of the articles retrieved.


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All RCTs with an adequate control group in which iron supply was provided by natural food sources, fortified foods, formula, or supplements until the age of 18 years were considered for inclusion. No language restrictions were applied. 13 supplementation studies met the selection criteria for neurodevelopment and 7 for immunity. Most of the studies had a high or moderate risk of bias. A large variety of outcomes were reported. Overall, the studies showed a modest positive effect of iron supplementation on cognition and psychomotor outcomes, especially in anemic infants and children and after long-term supplementation periods (>2 months). A comparison of studies using immunity outcome was not possible due to heterogeneity. Currently available evidence is too incomplete to make evidence-based conclusions on the effect of iron on immunity.

This research was undertaken as an activity of the European Micronutrient Recommendations Aligned (EURRECA) Network of Excellence (www.eurreca.org), funded by the European Commission Contract Number FP6 036196-2 (FOOD). The original conception of the systematic review was undertaken by the EURRECA Network and coordinated by partners based at Wageningen University (WU), the Netherlands and the University of East Anglia (UEA), United Kingdom.

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Influence of an iron fortified fruit juice on bone remodeling in women at risk of

iron deficiency anaemiaBlanco-Rojo R.1, Pérez-Granados A.M.1, De la Piedra C.2,

Vaquero M.P.1

1Department of Metabolism and Nutrition, Institute of Food Science and Technology and Nutrition (ICTAN), Spanish National Research Council (CSIC), Madrid, Spain; 2Laboratory of Biochemistry, Fundación Jimenez

Diaz, Madrid, Spain.

Introduction: Iron deficiency anaemia (IDA) is accompanied by a number of metabolic alterations. Severe IDA was related to deleterious effects on bone in rats, however, a clear association between bone and iron status has not been demonstrated in humans.

Objectives: To study the influence of consuming an iron fortified fruit juice, previously reported to be very efficacious at improving iron status, on bone biomarkers in menstruating women at risk of iron deficiency anaemia.

Methods: A randomised double-blind placebo-controlled study of 16-weeks of duration was performed. Inclusion criteria: menstruating women, ferritin<40ng/mL, hemoglobin≥11g/dL,


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18-45 years, non-smoker, non-pregnant, non-breastfeeding. Two groups: P group (n=18) or F group (n=23) consumed 500 mL/day of placebo fruit juice or iron-fortified fruit juice, respectively. The iron fortified-juice provided 18 mg of iron (100% of the RDA per day). At baseline, 8 and 16 weeks, iron and bone biochemical markers were determined: haemoglobin, serum ferritin, serum transferrin, 25-hidroxi-vitamin D (25OHD), alkaline phosphatase bone-isoenzyme (ALP) and serum cross-linked N-telopeptide of type I collagen (NTx).

Results: In F group, serum ferritin significantly increased (p<0.001) and serum transferrin significantly decreased (p<0.001) from baseline to week 16. No changes were observed in P group. Ferritin was significantly higher at week 16 with respect to P group (p< 0.01). Haemoglobin concentrations did not vary in P and F groups during the assay but at week 16 haemoglobin levels were significantly higher in F respect to P group (p< 0.01). No changes were observed in ALP and NTx and 25OHD significantly decreased in both groups (p<0.001).

Conclusion: Iron fortified fruit juice improves iron status and does not affect bone remodeling. The observed decrease in serum 25OHD in both groups clearly shows a seasonal effect.

Supported by GLPascual and Project Ref. AGL2009 11437/ALI. R.Blanco-Rojo was supported by a JAE-predoc grant from European Social Fund.

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Elucidation of the role of zinc, zinc-enzyme and tetrahydrobiopterin in the alteration of catecholamine metabolism

in the cerebral cortex with aging in senescence-accelerated mouse

Miyajima M.1, Minoshima M.1, Tanaka M.1, Nishimura R.1, Hosokawa T.2, Kurasaki M.3, Saito T.1,4

1Graduate School of Health Sciences, 2Institute for the Advancement of Higher Education,

3Faculty of Environmental Earth Sciences, 4Faculty of Health Sciences, Hokkaido University, Sapporo, Japan

The purpose in the present study was to elucidate contribution of catecholamines and zinc (Zn) to the decline of learning and memory with aging of senescence-accelerated mouse prone 10 (SAMP10). We determined concentrations of catecholamines, catecholamine metabolites and 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) act as a cofactor in catecholamine synthesis and the protein levels of enzymes in BH4 synthesis in the cerebral cortex.

We used male SAMP10 and SAMR1 (control) aged 12 month old. Catecholamines and their metabolites were determined by the HPLC-ECD. Zn concentration was determined with ICP-MS.


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We measured the protein levels of enzymes in BH4 synthesis by Western blot analysis. BH4 concentration was determined by HPLC with a spectrofluorometer.

The present study demonstrated decline of catecholamines in the cerebral cortex with aging of SAMP10, but no differences in concentrations of catecholamine metabolites between SAMP10 and SAMR1. These results suggested that the decline of catecholamines and Zn concentration were closely related to the dysfunction of learning and memory with aging of SAMP10, and the decline of catecholamines was caused by impairments of their synthetic pathway but not by enhancement of catabolic process. Moreover, there were no differences in the protein levels of tyrosine hydroxylase (TH) of rate-limiting enzyme in catecholamine synthetic pathway, GTP cyclohydrolase-1 (GTPCH-1) and 6-pyruvoyltetrahydropterin synthase (PTPS) enzymes in BH4 synthetic pathway between SAMP10 and SAMR1. But the protein level of sepiapterin reductase (SPR) and BH4 concentration in SAMP10 were significantly higher than in SAMR1. These results suggested that BH4 didn’t cause the decline of catecholamines but caused the senescence acceleration of SAMP10.

Session VI: Biofortification of trace elements

(Joint session with Cost Action FA0905)

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Biofortification of foods with macro- and micro-mineral elements

Martin R. BroadleyPlant and Crop Sciences Division, School of Biosciences, University of

Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK

Mineral malnutrition is widespread among the global poor. It can be addressed through dietary diversification, food fortification, supplementation, and by biofortification of edible crops, including fertiliser-based and crop-improvement strategies (White and Broadley, 2009). Even where food is abundant and diverse, dietary surveys reveal evidence of widespread mineral malnutrition, for example, significant numbers of UK and US adults consume calcium (Ca), magnesium (Mg), potassium (K) and selenium (Se) at quantities which are likely to be suboptimal (Broadley and White, 2010). In this talk, the potential for biofortification of staple and horticultural crops will be discussed using case studies in both developed and subsistence contexts. First, UK Se intakes and status have declined as milling wheat sourced from North America, whose soils are relatively high in Se, has been replaced with UK-grown wheat, whose soils are low in Se. Data from a industry/government project (2005-2009) will be presented to show the potential for using Se-containing fertilisers in a UK

Session VI: Biofortification of trace elements (Joint session with Cost Action FA0905)

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context, as practiced for >25 years in Finland (Broadley et al., 2010; Stroud et al., 2010). Second, new soil and grain survey from Malawi show that dietary Se intakes are extremely low for most of the population; experimental data again show that fertilisers have considerable potential for raising grain Se concentrations and dietary intakes at a national scale. Third, the impact of increased consumption and biofortification of edible horticultural products on dietary mineral intakes will be described.

References:

Broadley MR, Alcock J, Alford J, Cartwright P, Fairweather-Tait SJ, Foot I, Hart DJ, Hurst R, Knott P, McGrath SP, Meacham MC, Norman K, Mowat H, Scott P, Stroud JL, Tovey M, Tucker M, White PJ, Young SD, Zhao FJ. 2010. Selenium biofortification of high-yielding winter wheat (Triticum aestivum L.) by liquid or granular Se fertilisation. Plant & Soil 332: 5-18.

Broadley MR, White PJ. 2010. Eats roots and leaves. Can edible horticultural crops address dietary calcium (Ca), magnesium (Mg) and potassium (K) deficiencies in humans? Proceedings of the Nutrition Society 69: 601-612.

Stroud JL, Broadley MR, Foot I, Fairweather-Tait SJ, Hart DJ, Hurst R, Knott P, Mowat H, Norman K, Scott P, Tucker M, White PJ, McGrath SP, Zhao FJ. 2010. Soil factors affecting selenium concentration in wheat grain and the fate and speciation of Se fertilisers applied to soil. Plant & Soil 332: 19-30.

White PJ, Broadley MR. 2009. Biofortification of crops with seven mineral elements often lacking in human diets - iron, zinc, copper, calcium, magnesium, selenium and iodine. New Phytologist 182: 49-84.

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Zinc and iron biofortfication: harvestplus crop development update

Erick BoyHarvestPlus, Washington DC, USA

Developing more nutritious staple food crops (i.e. rice, wheat, beans, pearl millet) with high iron and zinc through biofortification requires combining high mineral content with characteristics that ensure acceptability by consumers, such as grain quality, high yield and pest and disease resistance. HarvestPlus plant breeding objectives for high iron and zinc for human nutrition include: 1) crop productivity equal or greater than conventional varieties; 2) stable genetic traits for high micronutrient levels across environments and climate conditions; 3) sufficient mineral concentration and bioavailability in the processed staple resulting in measurable impact on human nutritional status; and 4) organoleptic characteristics that are acceptable to consumers. Germplasm screening and field evaluation of commercial varieties and breeding lines of beans and pearl millet from multiple countries have produced encouraging results for achieving nutritionally desirable Fe increases of 45 μg/g and 30 μg/g, respectively. Increasing Zn concentrations in wheat and rice to nutritionally important levels is also feasible through


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traditional plant breeding. It seems that genetic engineering is necessary to effectively increase iron levels in rice and wheat. In vitro and animal models for screening varieties for Fe and Zn bioavailability add a useful dimension to the analysis of mineral concentrations. High Fe beans and pearl millet will be released respectively in Rwanda and India in 2011-2012.

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A defective ABC transporter of the MRP family, responsible of the bean low phytic acid (lpa1) mutation, affects the regulation of phytic acid pathway, reduces seed myo-

inositol and alters ABA sensitivityDario Panzeri1, Elena Cassani2, Enrico Doria3,

Giovanni Tagliabue1, Luca Forti4, Bruno Campion5, Roberto Bollini1, Charles A. Brearley6, Roberto Pilu2,

Erik Nielsen3, Francesca Sparvoli1

1Istituto di Biologia e Biotecnologia Agraria, CNR, Milano (Italy)2Dipartimento di Produzione Vegetale, Università degli Studi di Milano (Italy)

3Dipartimento di Genetica e Microbiologia, Università di Pavia, Pavia (Italy)4Dipartimento di Chimica, Università di Modena (Italy)

5Istituto Sperimentale per l’Orticoltura, CRA, Montanaso Lombardo, Lodi (Italy)6School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, (UK)

We had previously identified the lpa1 (low phytic acid) 280-10 line that carries a mutation conferring a 90% reduction of phytic acid (InsP6) content. In contrast to other lpa mutants, lpa1(280-10) does not display negative pleiotropic effects. We have now identified the mutated gene and analysed its impact on the phytic acid pathway.


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We mapped the lpa1(280-10) mutation by bulk analysis on a segregating F2 population, then, by comparison with the soybean genome we identified and sequenced a candidate gene. InsP6 pathway was analysed by gene expression and metabolites quantification. The mutated Pvmrp1(280-10) cosegregates with the lpa1(280-10) mutation, and the expression level of several genes of the InsP6 pathway are reduced in the lpa1(280-10) mutant as well as inositol and raffinosaccharides content. PvMrp2, a very similar paralog of PvMrp1 was also mapped and sequenced. The lpa1 mutation in bean is likely due to a defective Mrp1 gene (orthologous to the lpa genes AtMRP5 and ZmMRP4), while its Mrp2 paralog is not able to complement the mutant phenotype in the seed. This mutation appears to down-regulate the InsP6 pathway at transcriptional level, as well as altering inositol-related metabolism and affects ABA sensitivity.

Work is in progress aimed to evaluate the impact of the lpa1 mutation in plants exposed to drought stress or grown in phosphorus limiting conditions.

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Nutritional quality improvement in common beans bygenetic reduction of

phytates and other antinutritional factorsPerrone D.2, Doria E.3, Tava A.4, Dani V.4, Sparvoli F.5, Glahn R.6,

Nielsen E.3 and Campion B.1

1CRA – Unità di Ricerca per l’Orticoltura, Montanaso Lombardo, Lodi, Italy.2CRA – Centro di Ricerca per l’Orticoltura, Battipaglia, Salerno, Italy.

3Dipartimento di Genetica e Microbiologia, Università di Pavia, Pavia (Italy)4CRA - Centro di Ricerca per le Produzioni Foraggere e Lattiero-Casearie,

Lodi, Italy.5Istituto di Biologia e Biotecnologia Agraria, CNR, Milano, Italy.

6Robert Holley Center for Agriculture and Health,USDA, Ithaca, NY, U.S.A.

Abstract

When common bean grains are consumed, phyticacid, raffinosaccharides, polyphenols and tannins exert major antinutritional effects such as reduced phosphate, iron and zinc absorption, low protein digestibility, and flatulence. Moreover, if cooking is not properly carried out, lectins and protease inhibitorsmay exert toxicintestinal effects.

In order to improve the nutritional characteristics of bean grain used for human consumption and potentially for feedingmonogastric animals, we developedseveral innovative


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bean lines. First, we developed several lectin-free (lf) lines producing coloured seeds, then we combined the lf and the wsc(white seed coat, correlated with very low amounts of tanninsand polyphenols) traits in the same genetic background. Finally, we introduced the recently isolated low phytic acid (lpa280-10) trait in the lf and lf + wsc backgrounds.Since it is well known that lpa mutations may cause negative physiological effects in bean seeds, in particular lower seedling emergence and thus lower grain yield, we submitted the new bean lines to a field performance test carried out in two Italian locations, as well as to biochemical analyses and bioassays aimed to evaluate their nutritional and technological characteristics.

Obtained results were as follows:

1. The introgression of thelpa mutation caused large phytate reductions (80-90%) without affecting yield or introducingnegative agronomical effects.

2. Compared to a suitable control line, Fe bioavailability of bean flour (assayed by the in vitro digestion/Caco2 cell bioassay) was enhanced in a line with a low content of tannins and polyphenols and exhibited a further significant increase in a line with the same background additionally endowed with the low phytate trait.

3. Statistical analyses of the obtained biochemical data revealeda number of highly significant correlations between the 11 investigated parameters, some of which were expected (such as the positive interactions Fe/Zn, tannins/polyphenols), while others (for example the negative interactions protein/Pi, protein/tannins, protein/polyphenols) need further investigation to be interpreted.


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Moreover, unexpectedly, in the new beans we found a remakable increase of protein content.

Further testing and development of these breeding lines are underway to confirm the in vitro observations in an animal model.

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Model studies on zn-biofortification: modification of zn subcellular and

tissue distribution through heterologous expression of hma4 likely contributes to

the generation of the phenotype Siemianowski O.1 and Antosiewicz D.M.1

1University of Warsaw, Faculty of Biology, Institute of Plant Experimental Biology, Miecznikowa 1, 02-096 Warsaw, POLAND; [email protected];

[email protected]

A barrier in a root-to-shoot Zn-translocation is a major bottleneck in Zn biofortification. To overcome it, in previous studies we overexpressed AtHMA4 (P1B-ATPase known to control Zn flux to the shoots) in tobacco and enhanced Zn shoot level at 10 μM external Zn was detected. However, at high 100-200 μM Zn its concentration in leaves remained at the wild-type level, and surprisingly transgenic plants became more sensitive to zinc.

To clarify the mechanisms of the phenotype generation, we hypothesized that ectopic expression of AtHMA4 which is an export protein, may lead to overloading of the apoplast, consequently to the alterations of Zn distribution in tissues.


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As a first step, to verify this hypothesis we determined: (i) apoplast/symplast Zn partitioning by analysing Zn concentration in apoplastic fluids and protoplasts; (ii) Zn subcellular/cellular/tissue localization by Zinpyr-1 staining.

We showed that expression of AtHMA4: (i)increased Zn export form cells, (ii)caused Zn overloading of apoplasts; (iii)limited Zn accumulation in protoplasts. Moreover, we found modifications of Zn-tissue localization in root/leaf tissues. Interestingly in leaves of AtHMA4-transformants high Zn was detected in the apoplast and specifically inside and near necrotic regions, whereas in wild-type Zn was abundant inside numerous cells throughout leaf blades.

Results suggest that decreased Zn tolerance of AtHMA4-transformants result from higher Zn level in apoplasts what might induce an “alarm” response, and excess of Zn is directed to areas which ultimately develop necrosis protecting other parts of leaves from Zn toxicity. It seems that interplay between apoplastic and symplastic Zn concentration has an impact on Zn tolerance, also uptake and accumulation. We conclude that the expression of an export protein which is not tissue-specific leads to alterations of Zn-subcellular status which result in a modification of a plant “omics”, and may contribute to the development of an unexpected or even undesired plant’s phenotype.

Supported by: This work was financially supported by FP6 EU PHIME

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Size exclusion chromatography, coupled with inductively coupled plasma-mass

spectrometry is an effective method of screening cereals for potentially

bioavailable genotypes to aid future breeding strategies in tackling iron

deficency Tristan Eagling1, Fangjie Zhao1, Andy Neal1 , Steve McGrath1,

Peter Shewry1, Susan Fairweather-Tait2

1.Rothamsted Research Hertfordshire AL5 2JQ 2. Norwich Medical School, University of East Anglia, Norwich, Norfolk, NR4 7TJ, UK

Iron deficiency remains a prevalent nutritional disorder worldwide, disproportionally affecting people of low and middle income countries due to the reliance on non-meat sources in the diet. Cereals, a relatively poor Fe source, account for over 50% of the energy intake in developing countries, and are a potentially important target for biofortification strategies aimed at improving dietary Fe content and utilisation.

The effectiveness of a crop in combating Fe deficiency is largely dependent on Fe speciation, as different forms vary in their bioavailability. Size exclusion chromatography, coupled with


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inductively coupled plasma-mass spectrometry is a valuable approach to identify and quantify different forms of Fe in the grain. Adaptations of the method to quantify and identify nutritionally relevant forms of Fe (ferritin, Fe-phytate, mono-ferric phytate and Fe-nicotianamine) are described, together with the assessment of bioavailability of Fe in crops using an in vitro Caco-2 cell model system.

Nicotianamine synthase has been shown to increase Fe-nicotianamine concentration in both rice and wheat, and differences in Fe speciation profiles across tissues of both rice and wheat grain are presented. Differences in Fe speciation profile across genotypes have also been identified in wheat grain, which will aid future breeding strategies.

Funding: Tristan Eagling has a BBSRC studentship and additional funding is provided by HarvestPlus.

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Determination of heritability of zinc-efficiency in pepper (capsicum annum l.) And development of molecular markers

linked to qtl / genes controlling zinc-efficiency

Dr. Nedim Mutlu1, Dr. Davut Keles2, Ms. Hasan Pinar3, Ms. Atilla Ata3

1Akdeniz University, Faculty of Agriculture, Dept of Agricultural Biotechnology, Antalya, Turkey

2Alata Horticultural Research Institute, Erdemli-Mersin, Turkey3Alata Horticultural Research Institute, Erdemli-Mersin, Turkey (Ph.D student)

Zinc deficiency is a widespread problem reducing yield and quality of crop plants. Zn deficiency, estimated to cover a 14 million ha arable land, is widespread in Turkey. Zn deficiency causes stunting, and leaf chlorosis reducing yield and quality of crops due in part to loss in photosynthetic capacity. While considerable genetic variation exists to Zn deficiency (also known as Zn efficiency) in pepper (Capsicum annum L.) phenotypic selection is difficult and would benefit from the development of molecular markers. The objectives of this study are; 1. To elucidate the inheritance and heritabilities of genes and / or QTL


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determining Zinc-efficiency, and 2. To identify map locations of these genes / QTL in pepper. Populations derived from an interspesific (Capsicum frutescens x C. annum) cross will be used. The ultimate aim of this study is to determine the genetic basis of Zn efficiency in order to establish a method breeding for Zn efficiency. It is expected that hidden yield loss due to Zn deficiency can be alleviated. The parents (C.frutenscens PI 281420 and C. annum Alata 21), F1, F2, BC1P1 (backcross to C. frutescens) and BC1P2 (backcross to C. annum) will be utilized to determine the genetics behind zinc-efficiency in pepper. The F2 and F2-derived F3 families will be phenotyped and used to map the genes responsible for Zinc-efficiency. Phenotypic evaluations under zinc-deficiency, zinc contents of roots, shoots, and leaf data will be included to analyze both inheritance and map position of the genes / QTL that affect the zinc-efficiency in pepper. Zn efficiency of the plants will be recorded before they are harvested at the beginning of flowering. Dry weights and Zn-efficiency values, root, shoot, and leaf Zn contents of the parents, F1, F2, BC1P1 and BC1P2 will be used to estimate heritability and number of genes determining these characters under Zn-deficiency. For the mapping part of the study, 400 F2 plants along with parents will be grown under Zn deficient conditions, then after recording Zn efficiency (at the initiation of flowering), the plants will be switched to regular fertilization regime (+Zn) where F2 plants will be selfed to obtain F3 seeds. Based on Zn efficiency and leaf Zn content, selective genotyping will be performed among F2 plants. Among the 400 F2 plants, the most Zn efficient 60 F2, and the least Zn efficient 60 F2 plants will be identified and their F3 families (10 plants each of) will be grown under both Zn deficient and Zn-fertilized soils. The 94 F2 plants with consistent mean F3 family Zn efficiency, root, shoot, and leaf Zn content without any physiological disorders as observed within a given F3 family


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will be genotyped and used for mapping the genes / QTL for Zn efficiency. The co-dominant markers mapped on the integrated pepper map on a cross between ‘C. frutescens x C. annuum’ will mainly be used to locate and narrow down the location of genes / QTL that determine Zn efficiency. Data will be analyzed using Map manager QTX and QTL Cartographer. Results are expected to shed light to inheritance and chromosomal locations of genes / QTL for; 1.overall growth retardation under Zinc deficiency, 2. Zn uptake by roots, 3. Translocation of Zn from roots to shoots, 4. (If exist) translocation of Zn from shoots to leaves.

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Effects of foliar zinc fertilizer application on zinc density and bioavailability in

rice (Oryza sativa L.) Yanyan Wei1*, M. J. I. Shohag1, Xiao-e Yang1*, Yuyan Wang1,

Zhenli He2 and Peter J. Stoffella2 1MOE Key Laboratory of Environmental Remediation and Ecosystem

Health, College of Environmental and Resources Science, Zhejiang University, Hangzhou, 310029, P. R. China; 2Indian River Research and

Education Center, Institute of Food and Agricultural Sciences, University of Florida, Fort Pierce, FL34951-3138, USA.

[email protected] or [email protected]

Zinc (Zn) deficiency has been recognized as a global public health problem. Breeding to increase high amount and bioavailable zinc in rice grain has been suggested as a way to alleviate Zn malnutrition in human. The aims of this study were to assess the effect of foliar Zn fertilizers on zinc density in the polished rice under field condition, and Zn bioavailability in Caco-2 cells. Four rice cultivars with contrasting agronomic traits and five foliar zinc fertilizers (Zn-EDTA, Zn- Citrate, ZnSO4, Zn-amino acids) were adopted, and Caco-2 cells model were used to study the zinc bioavailability as the indicator. The results showed that foliar application of different zinc fertilizers can improve the zinc content in selected rice genotypes. Compared


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with the control, application of ZnSO4, Zn-AA, Zn-Citrate and Zn-EDTA increased grain Zn content by 17.8%, 17.2%, 9.3% and 4.0%, respectively. Large difference in Zn concentration among the four genotypes were noted, and CV-BY grain contained two times higher Zn than CV-LY9. Zn bioavailability measured as total uptake by Caco-2 cell The zinc fertilizer had no direct effect on iron, calcium, phytic acid, and protein content differed from rice genotypes and foliar fertilizer types. The dense cultivar like BY TN85 showed greater Zn uptake by Caco-cell, and Zn-AA application increased Zn bioavailability mostly, followed by ZnSO4. Whereas little effects was observed of application with Zn-citrate and Zn-EDTA on Zn bioavailability. Further study on biochemical compositions indicated that application of Zn-AA and ZnSO4, considerably decreased the phytic acid/zinc molar ratio, and increased sulfur, met, and cysteine concentrations in polished grain, especially for the dense cultivars (BY). The zinc bioavailability of the rice genotypes was related with the content of zinc, sulfur, lysine and cysteine, respectively. The results indicated that grain zinc density and bioavailability were affected by fertilizer type and genotypes, but genotype was the dominate factor. Application of foliar zinc fertilizer can improve the zinc density and bioavailability, and ZnSO4, Zn-AA are better sources than Zn-citrate and Zn-EDTA. The results suggest that higher zinc, sulfur, Met, and cysteine contents can be used for indicators for screening higher zinc bioavailable rice cultivar.

Keywords: Zinc, Foliar fertilization, Density, Bioavailability, Caco-2 cells.

Session VII: Trace element analysis and quality control

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Isotopic tools and metrology principles for reliable inorganic chemical

measurements – illustration with reference values for the EU Reference

Laboratory on heavy metals in feed and food

Christophe Quétel, Beatriz de la Calle, Agnieszka KrataEuropean Commission – Joint Research Center

Institute for Reference Materials and Measurements111, Retieseweg. B-2440 [email protected]

In European legislation measures are taken to control the level of exposure to contaminants and protect the public health. For instance, regarding undesirable substances present in animal feed materials and additives, because of natural or industrial reasons, the EC Directive 2002/32 (and amendment 2003/100/EC) fixes maximum concentrations permitted to prevent harmful effects on animals or consumers. And the EC Regulation 1881/2006 establishes similar types of limits in foodstuffs.


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The safety of citizens requires that measurement results used to produce information and take decisions are accurate and traceable to a stated system of reference (SI preferably). Traceability ensures comparability in time and space, from a measurement sector to the other, between organisations as well between countries. Accuracy depends on the competence of the chemist and the level of performance of the measurement method. Method validation is a central pillar of the ISO/IEC-17025 standard, and approaches described therein, such as the assessment of the uncertainty of the results and the participation to inter-laboratory comparisons, are potentially extremely powerful.

This presentation will provide examples of how isotopic methodology and these principles of metrology in chemistry may be associated to produce reference data on metal concentration and isotopic signatures in environmental and food samples. The EU Reference Laboratory on heavy metals in feed and food is operated at IRMM and works, in the frame of the above described legislation, together with appointed national reference laboratories of the EU Member States. Results of proficiency tests for these laboratories will be presented for the measurement of the Cd, Pb and Hg concentrations in animal feeds, when applying total digestion of the matrix and partial extraction with 5% HNO3. The way isotope dilution mass spectrometry can be implemented to deliver independent reference measurement results in both cases will be illustrated.

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Ultra-trace speciation by online preconcentration Liquid Chromatography

Inductively Coupled Plasma Mass Spectrometry to study the environmental

fate of elemental selenium Topic: Micronutrient Biofortification / Advances in Trace

Elements Analysis and Quality Control

Lenz, M.1), Floor, G.H.2,3), Evangelou, M.W.H4), Schulin, R.4), Philippe F.X. Corvini1

1. Institute for Ecopreneurship, University of Applied Sciences Northwestern Switzerland (FHNW). Gründenstrassse 40, CH-4132 Muttenz, Switzerland

2. Department of Chemistry. University of Girona. Campus de Montilivi s/n. 17071 Girona, Spain

3. Department of Meteoroloy, Monitonring and Analysis, BRGM. 3 avenue Claude-Guillemin BP 36009, 45060 Orléans Cedex 2, France.

4. Institute of Terrestrial Ecosystems, ETH Zürich, Universitaetstrasse 16, 8092 Zürich, Switzerland

* Corresponding /presenting author: [email protected]

Selenium deficiency is a problem of worldwide concern, affecting the health of 0.5 to 1 billion people [1]. Biofortification aims at increasing the nutritional value of forage plants grown on selenium deficient soils e.g. by applying appropriate fertilization strategies. However, only 5 to 30% of the selenium amended


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to mineral fertilizers in form of selenite and selenate salts is taken up by plants [2]. This inadequate uptake is partly due to leaching from soil or sorption to soil components, depending on the selenium speciation. Elemental selenium can provide plant-available selenium species (selenite, selenate) during its slow oxidation and thus promises to minimize leaching of selenium from soils when applied as fertilizer. It is evident that the study of relevant field concentrations (8 g Se / ha for grain, 3 g / ha for grass fields, [3]) demands for outstandingly sensitive methods for quantification of selenium species . This study used Liquid Chromatography Inductively Coupled Plasma Mass Spectrometry (LC-ICP-MS) to quantify selenite and selenite down to field relevant concentrations, i.e. the low ppt range. The high sensitivity was achieved by two complementary strategies. Firstly, selenium species were concentrated on an anion trap column that was coupled online to the LC-ICP-MS. Secondly, pressurizing the octopole reaction system with hydrogen allowed to use the more abundant selenium isotopes 78Se and 80Se instead of the less abundant 82Se. The chromatographic method furthermore separated bromine from selenium species, eliminating the potential 1H79Br interference when using hydrogen to remove interferences. The developed method was applied to investigate the kinetic of elemental selenium oxidation in field relevant concentrations. With this online preconcentration LC-ICP-MS at hand, it is now possible to study selenium speciation at ultra-trace concentrations, necessary for most selenium deficient natural environments.

Supported by: Swiss National Science Foundation (Grant Number SNF 200021-126899); COST Action FA0905 (Mineral-improved Crop Production for Healthy Food and Feed); FP6 AquaTRAIN Marie Curie Research Network (Contract No. MRTN-CT-2006-035420).


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References:

[1] Haug, A., R. D. Graham, et al. (2007). Microbial Ecology in Health and Disease 19(4): 209-228.

[2] Eich-Greatorex, S., T. A. Sogn, et al. (2007). Nutrient Cycling in Agroecosystems 79(3): 221-231.

[3] Mäkelä, A., W. Wang, et al. (1995). Biological Trace Element Research 47(1): 289-298.

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Capillary Electrophoresis coupled with an Inductively Coupled Plasma

mass Spectrometry detector permitted speciation and quantification of metal glycinate complexes in feed premixes

C. Ionescu*1, V. Vacchina2, R. Lobinski3 and D. Bravo1

1Pancosma, Geneva, Switzerland, 2UT2A, Pau, France, 3CNRS, Pau, France.

Trace elements inclusion as feed additives is based on their metal content. This is mainly due to the unavailability of analytical methods differentiating trace element sources.

The objective was to develop a method allowing specific determination of Zn, Cu, Mn glycinate complexes (BT) from the corresponding sulfate (SU), citrate (CI) or histidinate (HI), preserving BT molecular integrity and to check its application to premixes.

Capillary Electrophoresis (CE) coupled with Inductively Coupled Plasma mass Spectrometry (ICP-MS) gave the best results. BT Cu was well separated from SU, CI and HI. BT Zn was well separated from SU and HI but not from CI. BT Mn was well separated from


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SU but there was an overlapping with CI and HI. However, CI and HI were reconstructed complexes and not standards. BT molecular integrity was checked by CE coupled to electrospray tandem mass spectrometry.

Analytical figures of merit of the CE-ICP-MS coupling were determined. The calibration curve was linear (R2 > 0.995). The repeatability was below 12%. Accuracy was generally below 15%. Detection limits were between 0.05 and 0.2 μg metal.mL−1 depending on the BT.

The method was then used to quantify BT in four premixes based either on minerals (as sulfates), choline chloride, amino-acids or acid salts. The premixes were spiked with BT Cu (6.4 mg/g), BT Zn (8.9 mg/g) and BT Mn (18.7 mg/g). A single peak, absent from the blank sample and migrating at the same time than BT standard, was detected in the electropherograms of the premixes extracts. The BT concentrations measured experimentally (between 8.1 and 9.7 mg/g for BT Zn ; 6.6 and 7.2 mg/g for BT Cu and between 18.3 and 20.7 mg/g for BT Mn) were in agreement with the expected concentrations showing that BT can be analysed and quantified in premixes with the method developed.

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Gas chromatography coupled with atomic emission or mass spectrometry

detectors for the determination of organotin compounds in foods

N. Campillo, P. Viñas, R. Peñalver, J.I. Cacho and M. Hernández-Córdoba

Department of Analytical Chemistry, Faculty of Chemistry, University of Murcia, E-30100, Murcia, Spain. e-mail: [email protected]

Organotin compounds (OTCs) are widely used in diverse human activities, mainly in agriculture and industrial processes. Contamination of the environment by OTCs is a real problem and, due to their toxicity, the possible risk posed to humans by food intake is of a great concern.

Solid-phase microextraction conditions for the separation and preconcentration of seven OTCs [monobutyltin (MBT), dibutyltin (DBT), tributyltin (TBT), monophenyltin (MPhT), triphenyltin (TPhT), fenbutatin oxide (FBYO) and the sum of cyhexatin (CTC) and azocyclotin expressed as CTC] in foods (namely honey and wine samples) have been optimized. The samples were first derivatized in situ and then retained on a 100 μm PDMS fiber exposed to the headspace of solutions containing


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the food sample under analysis at 90 °C. Next the OTCs were separated using a capillary column containing 5% diphenyl- 95% dimethylpolysiloxane. Appropriate heating programs were applied in order the analytes to be eluted between 3.4 and 16 min. Two detection systems, atomic emission detector (AED) and mass spectrometry (MS) were evaluated. A matrix effect was observed for the sample matrices studied. To avoid the use of the standard additions method, and since diphenyltin proved to be absent in all the samples, this compound was used as an internal standard, which allowed the simplest, direct calibration against standard solutions to be carried out. The sensitivity was higher when MS was the detection system although AED provided simpler chromatograms. For the case of using MS, the detection limits ranged from 0.3 to 4.3 pg (Sn) g-1, for honey samples and from 0.1 to 2 pg (Sn) mL-1 for wine samples. No OTCs were detected in the commercial honey samples analyzed. However, MBT, DBT, TBT, TPhT and CTC were found in some wine samples at concentration levels in the 0.05-5 ng (Sn) mL-1 range.

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A diet for health and longevity. How do we get there?

Bruce N. Ames Senior Scientist, Nutrition and Metabolism Center Children’s Hospital

Oakland Research Institute Oakland, California Professor Emeritus, University of California, Berkeley

1) An inexpensive intervention could delay the degenerative diseases accompanying aging, such as cancer, cardiovascular disease, cognitive decline, and immune dysfunction. Most of the world’s population, even in developed countries, has inadequate intake of one or more micronutrients (~40 essential vitamins, minerals, fatty acids and amino acids) that a varied and balanced diet should provide. My triage theory (PNAS 103, 17589, 2006; AJCN 90, 889, 2009; M.A.D. 2010) posits that, as a result of recurrent shortages of micronutrients during evolution, natural selection developed a metabolic rebalancing response to shortage. The rebalancing favors micronutrient dependent proteins needed for short-term survival while starving those only required for long-term health. Triage theory predicts that the consequence of moderate shortages of even a single micronutrient, though insufficient to cause overt clinical symptoms, will impair functions essential for long-term health. This impairment will result in insidious damage (e.g. increased DNA damage) that,


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over time, leads to the acceleration of age associated diseases (e.g. increased cancer). As people with modest deficiencies have no overt clinical symptoms, there has been little incentive to correct these deficiencies, though this could change if it can be shown that they are resulting in biochemical changes, e.g. chromosome breaks, that are markers of increased risk of age-related diseases, e.g. cancer. The considerable experimental and theoretical support for the triage idea will be discussed as will a strategy for determining the optimum level of each micronutrient in humans. A perfect balanced diet (and adequate sunshine) would optimize levels of all micronutrients, but few reach this standard; fortunately inexpensive supplements and fortification can help. The triage theory should help to put micronutrient nutrition on a firm foundation and lead to preventive medicine for age-related diseases. 2) Too much refined food causes a shortage of micronutrients and fiber and an excess of calories (sugar, fat, and alcohol) which contributes to chronic inflammation, obesity, and associated diseases, such as diabetes. 3) How do we translate this new knowledge into improved health and lower costs?

Dr. Ames is a Professor of Biochemistry and Molecular Biology, Emeritus, University of California, Berkeley, and a Senior Scientist at Children’s Hospital Oakland Research Institute. He is a member of the National Academy of Sciences and he was on their Commission on Life Sciences. He was a member of the board of directors of the National Cancer Institute, the National Cancer Advisory Board, from 1976 to 1982. He was the recipient of the General Motors Cancer Research Foundation Prize (1983), the Tyler Environmental Prize (1985), the Gold Medal Award of the American Institute of Chemists (1991), the Glenn Foundation Award of the Gerontological Society of America (1992), the Honda Prize of the Honda Foundation, Japan (1996), the Japan Prize,


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(1997), the Medal of the City of Paris (1998), the U.S. National Medal of Science (1998), the Linus Pauling Institute Prize for Health Research (2001), the American Society for Microbiology Lifetime Achievement Award (2001), the Thomas Hunt Morgan Medal from the Genetics Society of America (2004), and the American Society for Nutrition/CRN M.S. Rose Award (2008). His 540+ publications have resulted in his being among the few hundred most-cited scientists (in all fields).

www.bruceames.org; [email protected]

Session VIII (Cost action FA 0905) homeostasis of trace elements in plants

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Mechanisms of arsenic transport and detoxification in plants

Fang-Jie Zhao Rothamsted Research, Harpenden, Herts AL5 2JQ, UK

Arsenic contamination occurs in the soils in many regions of the world, which may affect crop yield or result in excessive accumulation in food crops posing a health risk to humans. It is therefore important to understand how plants accumulate and tolerate As. The pathway of As uptake depends on its chemical species. Depending on the environmental conditions, As may be present as arsenate, arsenite or methylated As species. Arsenate is taken up by plants via the phosphate transport pathway. Evidence will be presented showing how mutation in phosphate transport systems or overexpression of certain phosphate transporter genes affects As uptake and translocation in rice in both hydroponic and soil experiments. In contrast to arsenate, arsenite uptake is mediated mainly by the silicon (Si) transport pathway in rice. The aquaporin channel Lsi1 (OsNIP2;1) mediates the influx of both silicic acid and arsenous acid into the root cells, whereas the carrier protein Lsi2 is responsible for the efflux of both Si and arsenite from the cells to the apoplast toward the stele. There is also evidence that OsNIP2;1 is


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involved in the uptake of methylated As species. Plants employ different mechanisms to detoxify As. In hyperaccumulators, detoxification involves arsenate reduction to arsenite which is then stored in the vacuoles. In nonhyperaccumulator plants, following arsenate reduction, arsenite is extruded to the external medium or is complexed by thiols and stored in the vacuoles. Efflux of arsenite to the external medium may be mediated by some aquaporin channels or unidentified mechanisms. Recently, vacuolar transporters for arsenite and arsenite-phytochelatin complexes have been identified. Uncomplexed arsenite and arsenate are the main forms of As transported in xylem, whereas the former dominates in the phloem exudate. Potential methods for reducing As accumulation in food crops will be discussed.

145

Translocation and biotransformation of applied selenium in brassica and accumulation of valuable selenium

compounds Seppänen M.M.1, Kontturi J.1, López Heras I.2, Madrid Y.2,

Cámara C.2, Hartikainen H.3

1) Department of Agricultural Sciences and 3) Department of Food and Environmental Sciences, 00014 University of Helsinki, Finland

2) Departamento de Química Analítica. Facultad de Ciencias Químicas. Universidad Complutense de Madrid. Avda Complutense s/n. 28040

Madrid, Spain

Selenium (Se), an essential micronutrient for humans and animals, is circulated to food chain via crop plants. Its deficiency can cause health problems, including muscle and cardiovascular disorders, immune and endocrine dysfunction, cancer and neurological problems. Agronomic biofortification with Se is used in areas where soil is low in bioavailable Se. Brassica species are efficient in Se uptake and therefore an attractive choice of species for biofortification. Selenium uptake, translocation and biotransformation in Brassica were studied with plants grown in field and greenhouse, and the accumulated Se species were identified and quantified by a HPLC-ICP-MS based method,


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using different separation chromatographic modes. Field-grown Brassica plants took 30 - 40% of the applied selenate at rosette stage but the translocation to seeds was low. Apparently, a significant loss of Se took place as the Se content of harvested seeds, plant residue or soil did not explain the fate of applied Se in field ecosystem. High concentration of Se in leaves was associated with a transcript accumulation of ATP sulfurylase (APS1–3) and selenocysteine methyltransferase (SMT) genes and transient accumulation of small amounts of Se-methylselenocysteine (SeMetSeCys). The most prominent Se species in leaves and seeds was selenomethionine (SeMet). In seeds SeMet is mainly located in the protein-rich meal fraction which is a valuable protein feed for animals. Agronomic biofortification with Se improves the nutritive quality of Brassica seeds due to high accumulation of SeMet which remains stable during seed processing. The Se cycle in field ecosystem still leaves open questions which are discussed.

147

Regulation of the plant zinc deficiency response

Ariadne Ribeiro Henriques1, Ana Carolina A.L. Campos1, Ya-Fen Lin1, Henk Schat2, Ana G.L. Assunção1#, Mark G.M. Aarts1

1Laboratory of Genetics, Wageningen University, Wageningen, The Netherlands; 2Department of Genetics, Molecular and Cellular Biology, Vrije

Universiteit, Amsterdam, The Netherlands;#current address: Department of Plant Biology and Biotechnology, University

of Copenhagen, DK-1871 Frederiksberg C, Denmark

Zinc is an essential micronutrient for all living organisms. When facing a shortage in zinc supply, plants adapt by enhancing the zinc uptake capacity. This involves the enhanced expression of a range of external metal uptake transporters and internal metal transporters, along with the enhanced expression of genes involved in metal chelation. We are interested in understanding the regulation of the zinc deficiency response.

Recently we used yeast-1-hybrid analysis to clone two transcription factors from Arabidopsis thaliana, bZIP19 and bZIP23, which are essential for the ability to respond to zinc deficiency. Along with these, we also identified the promoter cis-element to which these transcription factors bind, which consists of a 10-bp imperfect palindrome, often found with two copies in the target


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promoters, defining the list of direct target genes involved in Zn deficiency response. The availability of these transcription factors offers interesting options to modify the zinc deficiency response of plants, either by making them less sensitive to zinc deficiency, or by conferring a constitutive zinc deficiency response, which can induce plants to overaccumulate zinc. The results of our first experiments towards such applications will be presented and discussed, along with the functional analysis of some of the direct target genes.

This work is supported by the Netherlands Organisation for Scientific Research (NWO) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) of Brazil.

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Zn-/Fe-cross homeostasis involved in the generation of the Zn-related phenotype

of transgenic tomatoBarabasz A. and Antosiewicz D.M.

University of Warsaw, Faculty of Biology, Institute of Plant Experimental Biology, Miecznikowa 1, 02-096 Warsaw, POLAND;

[email protected]; [email protected]

Engineering of enhanced Zn uptake and accumulation in the shoots is one of the tool used in biofortification of plants in minerals. In tis study, an effort has been undertaken to increase Zn shoot / root ratio by overexpressing AhHMA4::AhHMA4 (P1B-ATPase involved in the control of Zn root-to-shoot translocation) in tomato. To test the effects of the AhHMA4-expression, transgenic and WT plants were exposed in hydroponics to a range of Zn concentrations (0-20μM). Plant performance, Zn/Fe accumulation, expression of AhHMA4 and chosen tomato endogenes were investigated.

AhHMA4 expression in tomato significantly enhanced Zn level in shoots at 10μM Zn supply but not at all Zn-treatments. Thus, Zn-supply dependent modification of Zn accumulation was detected. Transgenic plants, however, appeared to be more


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Zn-sensitive, and this trait was associated with reduced Fe root-to-shoot translocation. Expression analysis demonstrated that Fe-deficiency responsive genes involved in Fe/Zn uptake were upregulated (IRT1, FRO1 and FER) in transgenic tomato compared to WT level, as well as NRAMP1 involved in metal-redistribution. Moreover, differential expression of LeCHLN which depended on Zn-exposure was also detected. Further study showed that Zn apoplast concentration was much higher in transgenics, and was accompanied by higher expression level of LePMEU1 involved in cell-wall modification.

Results indicate the involvement of the alteration of Zn subcellular distribution and the modification of the expression of Fe-deficiency tomato responsive genes in the generation of Zn-related phenotype of transgenic tomato expressing AhHMA4.

Acknowledgements: This work was supported by FP6 EU PHIME project (FOOD-CT-2006-016253).

151

Lessons from Cd hyperaccumulatorsVerbruggen N.

Lab of Plant Physiology and Molecular Genetics, Université Libre de Bruxelles

Cadmium is one of the most toxic trace metallic elements for living organisms. Cadmium toxicity is due to its capacity to react with thiol groups of proteins and of glutathione and to compete with essential elements (1). Cadmium accumulation in soils is a world wide environmental problem and a threat for health.

A rare class of tolerant plants called hyperaccumulators are able to accumulate high foliar concentrations of cadmium without toxicity symptoms (2). Cadmium hyperaccumulators constitute fascinating study cases to understand detoxification mechanisms in plants. Furthermore study of hyperaccumulators can provide novel tools in phytoremediation and food security. Knowing the determinants of Cd tolerance and accumulation can help to develop plants able to stabilise or extract Cd in contaminated soils or to develop crops with limited Cd accumulation in the consumed parts.

Noccaea caerulescens and Arabidopsis halleri are both close relatives to A. thaliana, and the two most studied hyperaccumulator


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species up to now. While Zn hyperaccumulation seems to be constitutive in both species, Cd hyperaccumulation is confined to some populations.

Analyses of tolerance and accumulation have been realized on crosses between A. halleri and the non-tolerant non-accumulator species A. lyrata subsp. petraea. Three QTLs for Cd tolerance were identified, one co-locating with a QTL for Zn tolerance. Fine mapping showed co-localization of this common QTL with HMA4, encoding a Zn/Cd pump of the plasma membrane (3). Only one major QTL for Cd accumulation was identified, also co-locating with a QTL for Zn accumulation and co-localizing with HMA4, supporting that Cd tolerance and accumulation are not independent (4). HMA4 is highly expressed both in A. halleri and N. caerulescens. The importance of HMA4 in Zn hyperaccumulation and Cd hypertolerance in A. halleri was demonstrated using RNAi-mediated silencing (5). The accumulation of Cd was positively correlated with the accumulation of essential elements except Mg, which showed a negative correlation (4).

Transcriptomics studies have confirmed the importance of metal transport and detoxification processes in hyperaccumulation but have also shed light on other putative modifications of cellular processes (2). A review of possible players in Cd tolerance and accumulation will be presented.

References

(1) N. Verbruggen, C. Hermans and H. Schat Current Opinion in Plant Biology 12: 1-9 (2009)

(2) N. Verbruggen, C. Hermans and H. Schat New Phytologist 181: 759-776 (2009)


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(3) M. Courbot, G. Willems, P. Motte, S. Arvidsson, P. Saumitou-Laprade and N. Verbruggen Plant Physiology, 144: 1052-65 (2007)

(4) Willems G, Frérot H, Gennen J, Salis P, Saumitou-Laprade P and Verbruggen N New Phytologist vol 187 (2) 368-379 (2010)

(5) M. Hanikenne, I.N. Talke, M.J. Haydon, C. Lanz, A. Nolte, P. Motte, J. Kroymann, D. Weigel, U. Krämer Nature 453: 391-5 (2008)

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Poster P.1.1.

Effect of phytase addition on iron, cupper and zinc absorption in growing-finishing pig fed diets deficient in total

phosphorusLópez C., Madrid, J., Megías M.D., Martínez S., Hernández, F.

Department of Animal Production, University of Murcia, Murcia, Spain

Two experiments were carried out to study the effect of phytases on the apparent absorption of Fe, Cu and Zn for both growing and finishing pig. For this evaluation, nine crossbred barrows were used in each experiment. The initial weights were 47.8 and 71 kg for growing and finishing pig, respectively. Three experimental diets per phase were formulated: control diet containing 0.58 or 0.54 g P/kg (C), low P diet with 0.45 or 0.40 g P/kg (LP), for growing or finishing feed respectively, and low P diet with 500 phytase units/kg (LP+). Nine pigs were assigned to the 3 dietary treatments per phase according to 3 x 3 Latin square design. The diets were fed at a rate of 2.5 times the metabolizable energy requirement for maintenance. Each experimental period consisted of a 5-d adaptation period followed by a 7-d collection of feces.

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The content of Fe, Cu and Zn in diets was supplied from ingredients and mineral supplement. The average content of Fe, Cu and Zn on diets analyzed was 498, 20.8, 264 and 482, 18.5, 181 ppm for growing and finishing, respectively.

In growing phase, level of P in the diet affected to the apparent absorption of Fe, so it was increased (P<0.05) in lower-P total diets content (43.2, 56.5 and 58.4% for C, LP and LP+ diets respectively). The phytase addition increased (P<0,001) apparent absorption of the Cu in growing phase (-0.9, 10.2 and 34.7% for C, LP and LP+ diets respectively. More effects of phytase supplementation on mineral absorption were not observed. We concluded that phytase addition in the diet can increase Cu absorption in young pig.

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Poster P.1.2.

Effect of phytase addition in growing pig diets on tissue mineral concentration

López C., Madrid, J., Megías M.D., Valverde M., López M., Hernández, F.

Department of Animal Production, University of Murcia, Murcia, Spain

To determine the effect of the addition of microbial phytase on the Fe, Cu and Zn concentrations into liver, muscle, pancreas and bone, a 28-d experiment was conducted using 18 crossbred barrows (initial and final body weight of 31 and 54.5 kg, respectively).

Three experimental diets were formulated: control diet containing 0.58 g P/kg (C), low P diet with 0.45 g P/kg (LP) and low P diet with 500 phytase units/kg (LP+). The content of Fe, Cu and Zn in diets was 498, 20.8 and 264 ppm for C, LP and LP+ diets. The diets were fed ad libitum. At the end of experiment, pigs were sacrificed and liver, pancreas and muscle samples were taken off. Also III metacarpal bone from the left foot was removed and Fe, Cu and Zn concentrations in all tissues were analysed.

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The phytase addition decreased (P<0.05) the Fe concentration in muscle (83.8, 76.3 and 54.9 ppm for C, LP and LP+ diets respectively). In liver, a tendency (P=0.09) to decrease Fe concentration in tissue in LP diets was observed (226.6, 176.0 and 177.7 for C, LP and LP+ diets, respectively). The Cu concentration in liver, muscle, pancreas and bone were not affected by level of P or phytase addition to the diets. The Zn concentration in liver was increased (P<0.05) in LP diets (278.1, 375.7 and 297.5 ppm for C, LP and LP+ diets, respectively). However, in pancreas Zn concentration was alone increased (P<0.01) for phytase supplementation (200.2, 224.5 and 340.9 for C, LP and LP+ diets, respectively). In addition, Zn concentrations into muscle and bone were similar (P>0.05) between treatments. In conclusion, the lowering of phosphorus or/and supplementation with phytase in the diets for growing pig can change the Fe and Zn concentration in some tissues.

161

Poster P.1.3.

Determination of the coefficients of boron absorption from broiler diets

supplemented with boric acid

Arabela Elena Untea, Rodica Diana Criste, Margareta Olteanu, Gabriela Maria Cornescu,

Radu Corneliu Duca National Research and Development Institute for Biology and Animal

Nutrition (IBNA), 077015 Baloteşti, Calea Bucureşti, 1, jud. Ilfov, Balotesti, Romania, [email protected].

The role of trace elements is not fully ascertained in animal nutrition and the reports of the literature are contradictory. This is the case of boron (B) for which Hunt and Nielsen (1981), suggested a biological role of improving feeding efficiency and performance in broilers, besides alleviating the symptoms of vitamin D deficit. An experiment was conducted on 120 broilers (age 14d), 4 weeks, with the aim to determine the coefficients of boron absorption from the diets supplemented with boric acid. The broilers were assigned to 4 groups (C, E1, E2, E3), housed in metabolic cages (6 broilers / cage), and fed on a corn-soybean meal based diet. The experimental diets differed from the C diet

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as follows: E1 received 20 mg B / kg feed as boric acid; E2 received 20 mg B / kg feed as boric acid and 30% less vitamin D3 than group E1 and C (2000 IU vitamin D3 / kg); E3 received 10 mg B / kg feed as boric acid and vitamin D3 at the same level as E2. During the final 2 weeks of the experiment, for 5 days per week, the amount of given feed, the feed leftovers and the droppings were recorded with accuracy for each individual cage, as data for the mineral balance. The B concentrations were determined by DCP-AES in the samples (weekly samples/cage) of ingesta and faeces. The absorption coefficients of the dietary B were directly proportional to the dietary level: 27.79 % in C; 37.93% in E1; 41.37% in E2 and 38.54 % in E3. The deficit of vitamin D in the diet for E2 seems to have enhanced the absorption level. The supplemental boric acid didn’t influence the weight gain of the average daily gain of the experimental groups, compared to the control group.

Keywords: broilers, boron, boric acid, absorption, balance, vitamin D.

163

Poster P.2.1.

Effect of consuming zinc-fortified bread on serum zinc and iron status of

zinc-deficient womenNekouei N.1, Fazilati M.1 and Shahedi M.1

1Department of Food Science and Technology, Isfahan University of Technology, Isfahan, Iran

Abstract: After iron deficiency, zinc deficiency is a major micronutrient deficiency in developing countries, and staple food fortification is an effective strategy to prevent and improve it among at-risk-populations. No action has been taken to reduce zinc deficiency via flour fortification so far in Iran and little is known about the influence of zinc fortification of flour on zinc and iron status of blood serum and also about the optimum and effective amount of zinc compound that is used in food fortification. The objective of this study was to evaluate the influence of zinc-fortified breads consumption on zinc and iron status of blood serum. In the present study, three types of bread were prepared from non-fortified and fortified flours with 50 and 100 ppm elemental zinc in the form of sulfate. Eighty zinc-deficient women aged 19-49 years were recruited and assigned

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randomly into 3 groups; Volunteers received daily 1) One loaf of high-zinc bread, 2) One loaf of low-zinc bread, and 3) One loaf of non-fortified bread for one month. Serum zinc and iron were measured before and after the study. ANOVA, Duncan’s test, Paired t test and Food Processor were used for statistical analyses. Results showed significant increase of serum zinc and iron level in all groups (p<0.001) except in control one (p>0.05). Absorption of zinc and iron in the group consumed high-zinc bread was significantly greater than that in the group received low-zinc bread, (p<0.01) and (p<0.05) respectively. It is concluded that fortification of flour with 50-100 ppm zinc plays an important role in achieving adequate zinc intake and absorption in zinc-deficient people. It also appears that consuming zinc-fortified bread improves iron absorption.

Key Words: Bread fortification, Zinc sulfate, Serum zinc, Serum iron, Zinc deficiency

Supported by the chancellor of Isfahan Payame Noor University, Mohammad Fazilati.

165

Poster P.3.1.

Use of antioxidants against kidney damage caused by arsenic intoxication

during pregnancy and lactationPineda, J, Herrera, A, Antonio, M.T.

Department of Physiology (Animal Physiology II), Faculty of Biology, Complutense University, 28040 Madrid, Spain

Topic: Toxicology and risk assessment of trace elements.

Chronic exposure to arsenic (As) via drinking water is a worldwide public health problem. Once absorbed, the principal way of excretion is the urine, thus, kidney tends to accumulate more As and toxic effects may be harmful in this organ. Arsenic has been known to cause oxidative stress, it is also implies a possibility of preventing, at least partly, the ensuing toxicity by an increase of antioxidants. For this reason, the present study has been undertaken to evaluate the ameliorating effect of vitamin C, vitamin E and zinc on arsenic induced alterations in kidneys.

In our study, pregnant Wistar rats were divided into four groups: control group; arsenic group (50 mg/l of As); antioxidant group (vitamin C 2000 mg/l, vitamin E 500 mg/l, zinc 20 mg/l); and

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double treatment group(As + antioxidants). Experiments were performed in the kidneys of 21 days’ pups.

No changes were found in cholesterol, triglycerides and protein levels in the kidney of rats treated with As. A slight increase on relative weight and acid phosphatase activity in the kidney of this group occurred; it was not significant but it may suggest that animals might develop renal affectation.

Lipid peroxidation was estimated by thiobarbituric acid reaction (TBARS). It was 40% higher in the kidneys of As group than in control one. Combined administration of As + antioxidants reduced this to control values. Catalase is one of the most important enzymes implicated in detoxification processes; his activity was decreased (+ 20%) in the kidneys of As group with respect to the kidneys of control group; however, in As + antioxidant treatment did not return to normal values.

In conclusion, our results indicate that the combined administration of antioxidants could have an effective role reducing renal damage produced by As intoxication during foetal and neonatal development.

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Hepatic damage produced by low doses of arsenic intoxication during pregnancy

and lactation: antioxidants treatmentPineda, J., Herrera, A., Antonio, M.T.

Department of Physiology (Animal Physiology II), Faculty of Biology, Complutense University, 28040 Madrid, Spain

Topic: Toxicology and risk assessment of trace elements.

Arsenic (As) is considered the most common and hazardous xenobiotic in the environment. Several studies have demonstrated that liver is the primary arsenic metabolizing organ and, thus, an important target organ for its toxicity. There are few reports in which certain antioxidant compounds were used to repair the oxidative damage induced by arsenic; therefore, the present study aimed to investigate the protective role of antioxidants against arsenic-induced toxicity in liver of pups after gestational and lactational exposure to this element.

In our study, pregnant Wistar rats were divided into four groups: control group; As group (50 mg/L of As); antioxidant group (vitamin C 2000 mg/L, vitamin E 500 mg/L, zinc 20 mg/L); and

Poster P.3.2.

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double treatment group (As + antioxidants). Experiments were performed in the liver of 21 days’ pups.

Lipid peroxidation was estimated by thiobarbituric acid reaction (TBARS); it was 50% higher than control values in the liver of rats treated with As; combined administration of As + antioxidants reduced TBARS in the liver to control values. Catalase and Glutathione peroxidase (GPx) activities are possible mechanisms for defence against oxidative stress. In the liver of As group, catalase activity was three times lower than in control group (p < 0,01) while GPx activity had a slight increment. However, Antioxidant treatment was ineffective in returning the activity of catalase to normal values. Glutamic oxalacetic transaminase (GOT) activity was increased 10% in the liver of As group than in the liver of control group, indicating hepatic damage. This value returned to normal level in the liver of double treatment group.

To conclude, our results suggest that the administration of antioxidants could have an effective role reducing liver damage produced by As intoxication during foetal and neonatal development.

169

Analysis of heavy metals fromecological and non ecological

alcoholic beverages fromNorth West of Spain

Ordóñez, C., Cubría, J.C.Department of Biomedical Sciences University of León, Spain

The aim of our study was to determine the content of several heavy metals, of samples from ecological and non ecological slightly alcoholic beverages from North West of Spain, in order to evaluate whether the intakes of under study contaminants accomplished within recommended permissible levels.

Quantitative analysis of five toxic metals (Cr, Cu, Cd, Hg and Pb) were carried out by inductively coupled plasma quadrupole mass spectrometry (ICP-Q-MS). All obtained analytical results showed a good agreement at a 95% confidence level.

The obtained results shows the presence of metals in both, ecological and non-ecological products, these values were lower than limits given by official regulation. The levels for several

Poster P.3.3.

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metals (Cr, Cu, Cd, Hg and Pb) for a large number of samples are given. The detection limits of Cr, Cu, Cd, Hg and Pb were 1 mg/L, 0,5 mg/L, 0,1 mg/L ,0,5 mg/L and 0,3 mg/L. The dietary intakes of all five studied elements have also been estimated.

171

Cadmium daily intake from bean plants grown in tropical contaminated soils

Nogueira T.A.R.1, Abreu Junior C.H.1, He Z.L.2, Alleoni L.R.F.3, Trombeta A.L.B.3, Malavolta E.1†

1University of Sao Paulo (CENA/USP), Piracicaba, SP, Brazil. Email [email protected], [email protected]. †In memoriam.2Indian River Research and Education Center, Institute of Food and

Agricultural Sciences, University of Florida, 2199 S. Rock Road, Fort Pierce, FL 34945. Email [email protected]

3University of Sao Paulo (ESALQ/USP), Piracicaba, SP, Brazil. Email [email protected], [email protected]

Cadmium (Cd) is potentially toxic element to plants and humans. When present in food chain, it may cause serious damage in humans. This study was performed to estimate the daily intake of Cd from bean plants grown in tropical soils contaminated by Cd. The experiment was carried out under greenhouse conditions in Piracicaba, state of São Paulo, Brazil. Bean plants (three plants/pot) were cultivated in pots filled with 3 dm3 of two Oxisol (Typic Hapludox and Rhodic Hapludox) in a randomized block design in a factorial scheme (5 x 2) with three replicates. Treatments consisted of Cd rates added as CdCl2: 0.0, 0.5 (background value), 1.3 (alert value), 3.0 (intervention value), and 6.0 mg dm-3, based

Poster P.3.4.

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on the guideline established by the Environmental Agency of the State of Sao Paulo, Brazil - Cetesb. Daily Cd intake from bean was calculated by Cd content in bean grains multiplied with daily bean consumption (0.1; 0.2; 0.3; 0.4 and 0.5 kg, wet weight). Based on WHO/FAO provisional tolerable weekly intake (PTWI), fixed at 7 µg kg-1 of body weight for Cd, we calculated daily Cd intake taking into account the average of the human body weight (70 kg) to Brazilian population. Daily intakes of Cd from bean plants grown in the Rhodic Hapludox (for alert value) did not exceed the PTWI (ranging from 6.7 to 33.5 μg person-1 day-1). Conversely, in the Typic Hapludox (for alert value) the daily intake of Cd from bean plants was higher than the PTWI when the daily bean consumption was 0.2 kg. There is clear necessity to continuously monitor and to employ a surveillance system to ensure that the food supply and diet of the Brazilian population is safe.

Keywords: dietary risk, food contamination, human health, heavy metal

Supported by: this study was, in part, supported by a Ph.D graduate scholarship awarded to the first author (process # 07/59402-0) from FAPESP (The State of São Paulo Research Foundation) and by the University of Sao Paulo and University of Florida.

173

Poster P.3.5.

Arsenic, cadmium, lead and mercury content in foodstuffs of animal origin in

Finland in 2010Venäläinen Eija-Riitta

Finnish Food Safety Authority Evira, Helsinki, FinlandChemistry and Toxicology Unit

The national residue control programme is carried out annually in accordance with both national and European Union legislation. The aim of the programme is to assure that food is free from veterinary drugs, environmental contaminants and unauthorised or banned substances.

Heavy metals have been investigated in milk, eggs, honey and in meat, liver and kidney of bovine, pigs, poultry, horses, sheep, reindeer and elk. Mercury has been investigated in fish (rainbow trout). Samples were obtained from slaughterhouses or productions plants.

The Commission Regulations (EC) No 1881/2006 and No 629/2008 established the maximum levels (MLs) of cadmium, lead and mercury (in mg/kg wt) in certain foodstuffs.

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The average mercury content in investigated fish samples is <0.020 -0.030 mg/kg. The ML is 0.50 mg/kg.

Arsenic, cadmium and lead content in meat, honey, milk and eggs is low, <0.010 mg/kg. For cadmium in meat the ML is 0.050 mg/kg and for lead 0.10 mg/kg. For lead and cadmium in honey and eggs no ML values have been established by EU.

The average cadmium content in liver and kidney is below the admissible MLs (0.50 mg/kg for liver and 1.0 mg/kg for kidney), excluding the elk and reindeer. The cadmium content in liver (1.16 mg/kg) and kidney (6.45 mg/kg) of elk exceed the MLs given for bovine. Finnish legislation prohibits the use of liver and kidney of elks older than one year for human consumption. The cadmium content in liver (1.03 mg/kg) and kidney (3.28 mg/kg) of reindeer is also higher than the MLs.

The lead concentration in liver and kidney has decreased during the monitoring years and is now exceedingly below the admissible ML, 0.50 mg/kg.

Foodstuffs of animal origin produced in Finland contain very low levels of heavy metals except the high cadmium levels in internal organs of game animals.

175

Ultrastructural and microanalytical study of lactating mammary gland tissue after

terbium administrationAhlem Ayadi1, Samira Maghraoui1, Salem Abdessalem2,

Fedia Ben Hadj1 and Leila Tekaya1

1Laboratoire de Physiologie, Faculté de Médecine de Tunis. 15, Rue Djebel Lakhdar, La Rabta.1007. Tunis, Tunisie.

2Service de Cardiologie, Centre Hospitalo-Universitaire La Rabta. Tunis,Tunisie.

The frequent use of some lanthanides such as terbium (Tb) in medicine and industry makes us worry about their behaviour into the organism. Terbium is in the medical field as a radio-immuno-therapeutic agent to detect different forms of leukaemia, to determine binding sites of calcium in organs. Terbium is also used in the industrial domain such as television manufactures, batteries, computer’s screens, lasers,…

The aim of this work was to study the intracellular localization of terbium in the rat lactating mammary gland cells after its parenteral administration using transmission electron microscopy and ion mass spectrometry.

Poster P.3.6.

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Lactating female wistar rats received 7 intraperitoneal injections of a terbium soluble solution.

The ultrastructural and the microanalytical study was performed using respectively a Jeol 1010 transmission electron microscope and an IMS 6f ion mass spectrometer.

Ultrastructural observations showed the presence of many dense granules in the lysosomes of mammary glandular epithelial cells of the terbium treated rats. No deposit was observed in control rats

Ion mass spectrometry has demonstrated the presence of 159Tb in the lysosomes of the mammary glandular epithelial cells.

Previous studies established that the lysosomes of the liver, the kidney, the suprarenals.., concentrate actively mineral elements as an insoluble phosphate salt in the lysosomes of these large varieties of cells, after their administration as a soluble form.

The deposits observed in the cell lysosomes of the terbium treated rats are probably composed of an insoluble salt of terbium phosphate as demonstrated by the microanalytical studies.

These results demonstrated that the lysosome play an important role in the selective concentration-precipitation of mineral elements by their mixture of acid hydrolase which insolublilized foreign elements.

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Intracellular behaviour of indium and samarium in the lactating mammary

gland cells Samira Maghraoui1, Ahlem Ayadi1, Salem ABDESSALEM2, Fedia Ben Hadj1, Aouatef Ben Ammar3, Mohamed-Habib3

Jaaoura and Leila Tekaya1

1Laboratoire de Physiologie, Faculté de Médecine de Tunis. 15, Rue Djebel Lakhdar, La Rabta. 1007. Tunis, Tunisie.

2Service de Cardiologie, Centre Hospitalo-Universitaire La Rabta. Tunis, Tunisie.3Unité des Services Communs en Microscopie Electronique par

Transmission. Faculté de Médecine de Tunis. 15, Rue Djebel Lakhdar, La Rabta. 1007. Tunis, Tunisie.

Indium (IIIA group element) and samarium (lanthanide) are increasingly used in medicine and modern industry, reason for which many previous works have attempted to study their behaviour in many organs such as liver, kidney, intestine, etc. However, the precise intracellular localization of these two elements remains poorly understood in the lactating mammary gland cells.

We undertake, in this work, to compare the intracellular behaviour of indium and samarium in the lactating mammary gland cells.

Poster P.3.7.

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Lactating rats were given intrapritoneally a soluble solution of indium or samarium nitrate. Histological studies were performed using regular techniques of conventional transmission electron microscopy.

Our ultrastructural investigations demonstrated that the two studied elements have been concentrated in the glandular epithelial cell lysosomes. Nevertheless, a difference between the cellular consequences of the two group elements has been noticed. Many cellular damages have been observed in the glandular epithelial cells of the indium treated rats such as an expanded ergastoplasm and many altered mitochondria but no damage had been observed in the mammary gland cells of samarium treated rats. No deposit and no damage have been observed in the glandular epithelial cells of control rats.

IIIA group elements like indium, known for their high toxicity, seem to be more noxious than lanthanides. More sophisticated methods such as ion mass spectrometry and electron probe microanalysis have to be carried out to more understand the toxicity of these elements.

179

Poster P.3.8.

Reducing Cd accumulation in sweet pepper fruits by grafting

Morikawa C.K.1 and Saito T.1

1National Institute of Vegetable and Tea ScienceNational Agriculture and Food Research Organization

Tsu, Japan

Cadmium (Cd) level in sweet peppers ( Capsicum annuum L.) grown on contaminated soils could exceed the limits of 0.05g kg-1 established by the CODEX Alimentarius Commission of the United Nations Food and Agriculture Organization (FAO) and the World Health Organization (WHO). Two experiments were carried out in this study. One to select pepper rootstocks with low Cd content, and other to evaluate the capacity of the selected rootstock to reduce the Cd content of sweet pepper fruits by grafting. Eight pepper varieties were used in the screening experiment, and, the screening was made by measuring the Cd content of the aerial part of the plants at seedling stage. In both experiments, a highly contaminated soil (0.1N HCl 2.6 mg kg-1) was used. Experimental results showed that highly significant differences in Cd concentration were evident among eight pepper rootstock varieties. For Cd content, the order of pepper varieties

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was: Togarashi 4 > Piman Hi green > Kataguruma > Togarashi 5 > Suketto C > Belmasare > Belhomare > Daisuke. Daisuke variety was selected as low cadmium variety. The mean of cadmium contents of five fruits of grafted plants were significantly (P<0.05) lower than that of non-grafted one. The mean of Cd content of five fruits of non-grafted plants and grafted plants were of 0.09 and 0.05 mg kg-1 fresh fruit weight, respectively. Therefore we concluded that grafting onto Daisuke rootstock could be used to reduce the Cd content of sweet pepper fruits for human consumption.

Supported by: Ministry of Agriculture, Forestry and Fisheries of Japan (Research project for ensuring food safety from farm to table AC-1332).

181

Poster P.3.9.

Trace element toxicity from an abandoned mining site in a semiarid zone

García-Lorenzo, M.L.1, Pérez-Sirvent, C.1, Martínez-Sánchez, M.J.1, Molina-Ruiz, J.2

1Department of Agricultural Chemistry, Geology and Pedology.2Department of Geography. Faculty of Geography. University of Murcia,

Murcia (Spain). e-mail: [email protected]

Mining activities represent a current source of pollution, since trace elements are introduced into the atmospheric, terrestrial and aquatic ecosystems.

Determination of the contaminant content is not enough to evaluate the toxic effects or to characterise contaminated sites. Then, in order to estimate the risk of contaminants, chemical methods need to be complemented with biological methods. The aim of this study was to identify contaminants, hydric dispersion ways and reception areas of trace elements in the abandoned mining district of Sierra Minera Cartagena-La Unión and its influence area. In addition, the application of a bioassay battery has allowed confirming results, as well as the validation of the established conceptual model.

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The applied bioassays were: the bioluminescence inhibition of Vibrio fischeri in superficial water samples using Microtox® bioassay; the root and shoot elongation inhibition and the mortality of Lepidium sativum, Sorghum saccharatum and Sinapis alba using Phytotoxkit® bioassay; and inhibition of Heterocypris incongruens by way of Ostracodtoxkit®.

Obtained results suggested that mining activities in the studied area have produced great amount of wastes, characterized by high trace element content, acidic pH and minerals from supergene alteration. These materials are transported by torrential rainfall, an usual phenomenon in the studied area. During transport, neutralization processes acquire importance, favouring natural attenuation of trace element contamination by mixing with surrounding materials.

According to our results it is highly advisable to complement chemical analyses with environmental toxicity testing to characterise the risks presented by contaminated sediments. As regards the obtained results, the application of one bioassay does not discriminate all the contamination sources. However, the application of a test battery allows the identification of contamination sources and non contaminated areas, corresponding to decreasing toxicity values. Finally, these methods satisfy the requirements of environmental toxicology in their reliability, sensitivity, reproducibility, rapidity and low cost.

183

Poster P.3.10.

Utilisation of Zn chelate with amino acids in layer diets to reduce Zn excretion

in droppingsCriste Rodica Diana, Untea Arabela, Cornescu Gabriela Maria,

Bercaru Anca, Iulia MarosNational Research and Development Institute for Biology and Animal Nutrition(IBNA-Balotesti), 077015 Baloteşti, Calea Bucureşti nr. 1, Balotesti, Romania

The purpose of the experiment was to test a feeding solution with the purpose to reduce Zn excretion in layer droppings. The trial involved 96 Lohmann Brown layers aged 29 weeks, for 12 weeks, treated with a chelate (B-TRAXIMRTEC Zn-I30). The layers were housed in cages (3 layers/cage) and were assigned to 4 groups which received the same basic diet with 42.7% corn and 20% soybean meal. The premix of the control group (C) contained 60 ppm Zn (from zinc oxide), which is much over the requirement for layers (33 ppm, NRC, 1994). The supplemental level of Zn added to the experimental groups was 33 ppm (from zinc oxide) to E1, 33 ppm (from Zn chelate) to E2, 16.5 ppm (from zinc oxide) plus 16.5 ppm (from Zn chelate) to E3. The feeds were manufactured every other three weeks, throughout the

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experimental period; during the second week of feeding with a new batch of feed, all droppings were collected on a daily basis, and they formed average weekly samples/cage, from which Zn was determined by FAAS. A good correlation was determined between the ingested and excreted amounts of Zn (R= 0.87). The amount of Zn ingested by the experimental groups was about 33% lower than the amount ingested by group C. Under these conditions, the experimental groups had lower excretions of Zn: 9.01 mg/layer/day for E1; 8.44 (p≤ 0.05) for E2 and 8.39 (p≤ 0.05) for E3, compared to the excretion of Zn by group C (10.13 mg/layer/day). In the groups with Zn chelate (E2, E3), Zn excreta through droppings was more than 16% lower than in the control group. The study also determined the evolution of the bioproductive parameters (feed intake, weight gain, egg production).

Keywords: layers, Zn chelate, requirements, droppings.

185

Poster P.3.11.

The effect of organic and mineral fertilizers on the contents of metals and polyphenols; case study: grasses from

permanent grasslandHĂRMANESCU Monica1, GERGEN Iosif2

1. Banat’s University of Agricultural Sciences and Veterinary Medicine, Faculty of Agriculture, Calea Aradului 119, Timişoara, Romania,

[email protected]. Banat’s University of Agricultural Sciences Aand Veterinary Medicine,

Faculty of Food Technology, Calea Aradului 119, Timişoara, Romania, [email protected]

The goal of our study was to analyze the influence of mineral and organic fertilization on the heavy metals and total polyphenols contents in grasses from permanent grassland situated in a hill area in Banat County, Romania. Knowledge of these bioactive compounds in fodder grass is very important both for the nutritional or toxic effects in the plants or animals.

Experiment was conducted in 10 trials: 1 no fertilizers, 3 only with organic fertilizers, 3 with mixed fertilizers and 3 only with mineral fertilizers. All metals have been analyzed using flame

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atomic absorption spectrometry and Folin-Ciocalteu method was used for the total polyphenols content analysis. Statistica 6 and Unscramble 9 soft were used for Principal Component Analysis (PCA) and plot the data.

The correlations between metals and polyphenols contents and mineral or organic fertilizers are discussed in terms of PCA. Three factors can explain more than 86% from total variance of the model. PC1 is a complex principal component composed of polyphenols, Fe and Ni which correlate negatively with organic fertilizers, and Cu, Pb and Cd which correlate positively with organic fertilizers. Mn, Zn and Cd build the PC2. Mn is negatively correlated with organic fertilizers and positively correlated with mineral fertilizers; Zn and Cd are strongly positively correlated with organic fertilizers. Ni and Pb are the major component of PC3 but it has a small influence on the model.

Both fertilizers type influence the metal and polyphenols contents of the grasses, but in different manner. Generally, organic fertilizer increases the content of Zn, Cu, Pb and Cd, and mineral fertilizer in higher dozes decreases the content of metals (except Mn) and polyphenols. Principal Component Analysis can be an important tool for understanding the complex phenomena in biological systems.

Funding of the study: CNCSIS Romania, grant PD 576/2010 and grant nr. 52157/2008.

187

Poster P.3.12.

Determination of total arsenic in marine sediments, waters and fish tissues by

atomic fluorescence spectrometry1Martínez Sánchez M.J., 2González Párraga P., 2Cuesta A.,

2Esteban M.A., 1Martínez S., 1Pérez-Sirvent C.1 Department of Agricultural Chemistry, Geology and Pedology.

University of Murcia2 Department of Cellular Biology, University of Murcia.

e-mail: [email protected]

The importance of studying the As content of fish lies in the fact that this food seems to be one of the main ways in which the compound enters the human diet.

The aim of this work was to determine the concentration of arsenic in the different components that make up the marine ecosystem and to assess the possibility of it entering the food chain. For this purpose, the concentrations of arsenic were measured in waters, marine sediments and tissues (liver and muscle) of a widely eaten fish species (Sparus aurata).

The pH and electrical conductivity were determined in soil and water samples. The mineralogical composition in soil samples

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was studied by X-Ray diffraction (XRD), using a Philips PW3040 diffractometer and the Cu-Kα.

The arsenic concentrations in samples studied were determined using atomic fluorescence spectrometry with an automated continuous flow hydride generation (HG-AFS) spectrometer (PSA Millenium Excalibur 10055).

According to Cornelis et al., 2005, As concentrations of water and marine sediments are normally in a range of 1-2 μg/L and 3-15 mg/kg, respectively, and in our samples they were 1.30 μg/L and 2.85 mg/kg, respectively.

Many countries have set limits for the concentration of As permitted in fish; in Australia, for example, the limit is 1 mg/kg. In our study of gilthead seabream, 80% of muscle samples had concentrations below the limit of quantification, while the remaining 20% had a mean concentration of 0.21 mg/kg. In the liver samples analysed, 50% had concentrations below the limit of quantification and the other 50% a mean concentration of 0.18 mg/kg. In view of these results, Sparus aurata does no present a health risk and is not a significant factor in As entering the food chain.

189

Bioaccessibility of trace elements in waste mining materials, SE Spain

Martínez-Sánchez, M.J., Pérez-Sirvent, C., García-Lorenzo, M.L., González-Ciudad, E., Mantilla, W.,

Martínez-López, S., Martínez, L.B., Pérez-Espinosa, V.Department of Agricultural Chemistry, Geology and Pedology, Faculty of

Chemistry, University of Murcia, Murcia (Spain).e-mail: [email protected]

A study on metal mobilization and analysis of the health risk represented by ingestion (Zn, Pb, Cd and As) from contaminated sediments in Sierra Minera (SE Spain) was carried out. This zone has suffered a great impact from mining activities. The long-term deposition of metals in soils can lead to their accumulation and transport, while their toxicity depends on the mobility and bioavailability of a significant fraction of the metals. The ingestion of contaminated soil particles by animals or young children may represent a special exposure pathway for trace elements. Trace element bioavailability, rather than total content, is increasingly used as a key indicator of the potential risk that contaminants pose to both environmental and human health.

General analytical determinations (pH, particle size, organic matter, equivalent calcium carbonate content and mineralogical

Poster P.3.13.

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composition) were carried out in order to characterise sediment samples. An in vitro method for providing information on trace element bioaccessibility (Zn, Cd, Pb, Cu and As) in mine waste materials was also carried out.

Obtained results showed that the metal fraction dissolved by the proposed methodology is lower than 100%, both in the stomach and intestinal phases. Trace element solubility depends on its chemical speciation and binding capacity to different soil and sediment materials. Mineral associations, different alteration states and sorption/desorption processes play an important role in the gastrointestinal bioavailavility of metals.

Obtained results could be incorporated to the general protocol of risk assessment by ingestion applied to contaminated sites. This could be of interest since when risk assessments are adjusted to account for lower site-specific bioavailability, the resulting increase in cleanup levels can substantially reduce the cost of remediation in some cases.

191

Poster P.3.14.

Mechanism of toxicity on human vascular endothelial cells by humic acid

obtained from tropical peatlandKihara Y.1, Komori M.4, Sun Y.1, Sikder M.T.1, Hosokawa T.2,

Saito T.3, Kurasaki M.1, 4

1Graduate School of Environmental Sciences, 2Institute for the Advancement of Higher Education, 3Faculty of Health Sciences, Hokkaido University,

4Faculty of Environmental Earth Sciences, Sapporo, Japan

Humic acid, a group of high molecular weight organic compounds, is normally found in natural water, and characterized by an ability to complex heavy metals. Blackfoot disease is known as a health hazard by oral intake of humic acid. Although it has been reported the impairment to vascular endothelial cells at an early stage, the mechanism responsible for the disease is still less understood.

Humic acid in hydrosphere exists abundantly in the tropic peatland. Although humic acid rich water is used as drinking water for habitants at a peatland in developing countries, there is little knowledge about effects of it on human health.

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In this study, to clarify the effects of humic acid on human health, the cytotoxicity and the mechanism of the toxicity caused by the humic acid were investigated using of human vascular endothelial cells. The humic acid used in this study was purified from peatland in Central Kalimantan, Indonesia.

As results, humic acid obtained from Indonesia showed cytotoxicity. Since the expression of caspase 6 and 9 increased in the endothelial cells by humic acid administration, the cytotoxicity caused by humic acid was dependent on apoptosis. In addition, when oxidative stress was added to cell exposed to humic acid, the cytotoxicity was enhanced. And the cytotoxity was decreased by addition of vitamin C. From the results, the apoptosis induced by humic acid was owing to the increasing of oxidative stress. Furthermore, from the results that humic acid used contained iron, an iron chelator, DFO showed a decrease of humic acid-induced cytotoxicity, and addition of hydrogen peroxide with humic acid enhanced cytotoxicity, it was suggested that iron bound to humic acid mediate humic acid-induced cytotoxity.

193

Whole amaranth flour: evaluation of nutritive value as breadmaking

ingredientSanz-Penella, J.M. and Haros, M.*

Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC),Av. Agustín Escardino 7. Parque Científico, 46980 Paterna-Valencia, Spain,

*e-mail: [email protected]

In general, white bread has a low mineral content and should be supplemented to meet the daily requirements for different elements. Whole grain breads are known to be richer sources of macro- and microelements than breads made of refined flours. The purpose of the present work was to provide further information on how replacing wheat flour by whole amaranth flour from Amaranthus cruentus affects the phytate content of bread, and to evaluate its potential utility as a nutritious breadmaking ingredient. The mineral content increased significantly as a result of the replacement of wheat flour with whole amaranth, owing to the flour composition. The contributions of mineral intake from bread with or without amaranth to the dietary reference intakes (DRIs) given by the National Academy of Sciences, taking into account the World Health Organization’s recommendation

Poster P.4.1.

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of a daily intake of 250 g of bread per person were analysed. It is known that the bioavailability of minerals depends on the presence of phytic acid or phytate (InsP6), which acts as inhibitor of mineral uptake and have adverse effects on their bioavailability, owing to the formation of insoluble complexes. In this context, it is assumed that the predicted intakes that are derived from DRIs for the minerals analysed in this study are almost certainly overestimated. The amount of phytates and lower myo-inositol phosphates increased as the whole amaranth flour was introduced in the formulation. The phytate/minerals molar ratios are used to predict the inhibitory effect of InsP6 on the bioavailability of minerals. The high amounts of phytates found in amaranth flour, contributed to high levels in breads with amaranth, which could affect mineral bioavailability.

Supported by Consolider Fun-C-Food CSD2007-00063 Project from Ministry of Sciences and Innovation (MICINN), Spain

195

Poster P.4.2.

Effect of α-lactalbumin and nucleotides on calcium, iron and zinc uptake and

transport in Caco-2 cells from standard solutions

Peso Echarri P., Frontela Saseta C., González Bermúdez C., García Alonso J., González Marín A., Ros Berruezo G.,

Martínez Graciá C.Department of Food Science and Nutrition, Faculty of Veterinary Science,

Murcia University, Campus de Espinardo 30071, Murcia, Spain

Alpha-lactalbumin and nucleotides are ingredients that are added to infant formulas to mimic the human milk composition. Many beneficial effects have been reported in infants for these ingredients. Dietary nucleotides are nonprotein nitrogenous compounds that are important for growth, repair and differentiation of the gastrointestinal tract, and also could have a possible effect over mineral absorption. Few studies have been developed about this activity. The α-lactalbumin is a whey protein involved in many functional activities. Lactalbumin peptides obtained from α-lactalbumin in vitro digestion have been reported to act as mineral chelators through specific

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and nonspecific bindings sites. Therefore these peptides may be involved in mineral bioavailability. The objective of our study was to evaluate the mineral availability of calcium, iron and zinc from different standard solutions with or without α-lactalbumin or nucleotides by Caco-2 cultures. Caco-2 cells were seeded in filter of polyester membrane chamber inserts until its differentiation, at 19 to 21 days after seeding. Stock solutions contained aqueous 1M CaCl2 or 10 mM FeCl3 · 6H2O and 20 mM nitrilotriacetic acid (NTA) or 1mM ZnSO4 · 7H2O. Appropriate volumes of the stock solutions were added to the uptake buffer to obtain the following final concentrations: 10 and 30 mM of CaCl2, 50 y 150μM of FeCl3 · 6H2O, 5 y 10 μM of ZnSO4 · 7H2O which were added to the cells. The transport and uptake (retention plus transport) of calcium, iron and zinc were studied. Although without statistically significant differences, our results showed higher calcium uptake percentages with the addition of nucleotides. On the other hand, small statistically significant differences were found for α-lactabumin addition. Regarding to iron no statistically significant differences were found for cell uptake percentages. In the case of zinc, the availability could not be evaluated because of the zinc content from cells added with standard solution was similar to the zinc content from cells treated with the uptake buffer (blank).

This work was funded by the Spanish Plan Nacional de I+D+i through projects Consolider Ingenio 2010 Programme (ref. FUN-C-FOOD CSD2007-0623) and AGL-2007-63504.Thanks also to Fundación Seneca for the fellowship of P. Peso-Echarri (07877/BPS/07)

197

Poster P.4.3.

Influence of different caseinophosphopeptides on ferritin

synthesis in Caco-2 cellsGarcía-Nebot1 M.J., Alegría1 A., Barberá1* R. & Bouhallab2 S.

1Nutrition and Food Chemistry. Faculty of Pharmacy, University of Valencia, Avda. Vicente Andrés Estellés s/n, 46100 – Burjassot (Valencia). Spain.

2UMR1253, INRA, Agrocampus-ouest, Science & Technology of Milk and Egg (France).

* Corresponding author: [email protected]

Introduction: Iron deficiency is one of the most important nutrient deficiencies, and its more severe stages are associated with anemia being an effective strategy for preventing its deficiency the iron fortification. In this sense, caseinophosphopeptides (CPPs) are bioactive peptides derived from milk casein that can be released by enzymatic hydrolysis, gastrointestinal digestion, or during food processing. They present a structural cluster containing a sequence of three phosphoseryls residues followed by two glutamic acid residues that constitute the binding sites for iron, and thus play an important role in iron bioavailability.

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Objectives: To evaluate the influence of different caseinophosphopeptides on ferritin synthesis, as marker for iron uptake, in Caco-2 cells.

Methods: Samples: β(1-25) purified from a tryptic hydrolysate of bovine β-casein, two synthetic phosphopeptides (αs1(64-74) and αs2(1-19)), and two pools of CPPs obtained from β-casein and α-casein (commercial products) subjected both to simulated gastrointestinal digestion and selective precipitation (β-CPPs and α-CPPs, respectively). Complexes between iron (50 mM) and the different CPPs (12.5 mM) were performed. The ferritin synthesis in Caco-2 cells was determined via an enzyme-immunoassay (ELISA). The results were analyzed by one-way ANOVA and Tukey test to determine significant differences (p<0.05).

Results: The iron bound to specific CPP (β(1-25), αs1(64-74) and αs2(1-19)) (which contain the cluster) increased the ferritin synthesis (3.5, 2.6 and 2.5-fold, respectively) versus iron sulphate, β(1-25)-Fe being the most effective complex. The complexes between iron and β-CPPs or α-CPPs (CPPs obtained from β-casein or α-casein) did not improve the ferritin synthesis versus iron sulphate.

Conclusion: The results allow conclude that the efficiency of the CPPs on iron bioavailability (ferritin synthesis) depends of their structural properties being more effective the specific CPP than pool of CPPs (β-CPPs or α-CPPs).

This study has been partially funded by the CONSOLIDER INGENIO 2010 Programme, FUN-C-FOOD CSD2007-063. María José García-Nebot is the holder of a grant from the Generalitat Valenciana (Spain).

199

Poster P.4.4.

Investigations on factors impacting iron bioavailability from potato

Lisa Miranda, Christian Iammarino, Johanna Ziebel and Danièle Evers

Centre de Recherche Public-Gabriel Lippmann, Department EVA, 41, rue du Brill, L-4422 Luxembourg; [email protected]

Dietary iron deficiency is the most common nutritional disorder in the world. Increasing the amount of bioavailable dietary iron could prevent iron deficiency and anaemia. Potato is one of the most important crops worldwide as it is highly consumed. It contains high amounts of carbohydrates, ascorbic acid and polyphenols, among others. Moreover, it has been described to be a good source of minerals, such as potassium and magnesium; some cultivars have been shown to be rich in iron and zinc representing thus a good source for these micronutrients in human nutrition. The aim of this work is to evaluate iron bioavailability for human consumption from different potato cultivars and to study the impact of enhancers of iron bioavailability (e.g. vitamin C) or inhibitors commonly present in potato, such as chlorogenic acid, one of the major phenolic compounds. The final step is

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intended to study iron uptake from in vitro digested potato in relation to enhancers and inhibitors present.

In our studies, we used Caco-2 cells that acquire many characteristics from enterocytes during differentiation and that have been used in several studies on intestinal iron uptake and transport.

Preliminary ICP-MS and western blot results on Caco-2 cells show that Fe (II) and Fe (III) have different uptake mechanisms. Fe(II) is absorbed with almost no influence of vitamin C while Fe(III) uptake is influenced by vitamin C. Chlorogenic acid, inhibits Fe(III) uptake induced by the presence of vitamin C and inhibits Fe(II) uptake in presence or not of vitamin C. The results point to the complexity of potato, a potential source of iron in human nutrition, containing both enhancers and inhibitors of iron bioavailability.

201

Poster P.5.1.

Potassium apigenin: a promising radiation countermeasure

(1)Miguel Alcaraz; (4)Julián Castillo; (4)Obdulio Benavente García; (2)David Armero; (1)Miguel Alcaraz-Saura; (5)(*)Daniel Achel;

(3Vicente Vicente and (6)Manuel de las HerasDepartments 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.(6)Department of Radiotherapy. Clinic Hospital of San Carlos. University

Complutense of Madrid. C/ Prof. Martín Lagos s/n, 28040-Madrid.

The genoprotective and radioprotective effects of potassium apigenin against chromosomal damage induced by ionizing radiation were compared with those of L-ascorbic acid, δ-tocopherol, diosmin, rutin and the S-containing compounds (dimethyl sulfoxide (DMSO) and Amifostine), using the micronucleus test for antimutagenic activity to evaluate the reduction in the frequency of micronuclei (MN) in cytokinesis-blocked cells of human lymphocytes before and after irradiation; and the cell survival using the cell viability test with MTT in

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two prostate cell lines (PNT2, TRAMP-C1) administered before exposure to different X ray doses (0Gy, 4Gy, 6Gy, 8Gy and 10Gy).

The results point to the significant genoprotective capacity of the substances assayed before radiation: Apigenin (AP) = Diosmin (D) = α-tocopherol (E) = L-ascorbic acid (C) = Amifostine (AMIF) (p<0,001)> Rutin (R) = Dimetil sulfoxide (DMSO) (p<0,05)> Irradiated Controls (Ci), the maximum protection factor attained being 50%. When the substances were 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%. In contrast, AMIF, R and DMSO lost their genoprotective effect.

Cell survival obtained with 20 µM and 40 µM potassium apigenin administered before radiation with up to 10 Gy showed a Protection Factor of 100%, reducing radioinduced cell death by 30.8% in both cell lines assayed (p<0.001). Apigenin showed the greatest pre-treatment protective effect, a good post-treatment protection capacity and no toxicity towards cells.

For this reason, our results indicate that apigenin may be developed as a radioprotectant for humans against the potentially damaging effects of exposure to radiation. Human clinical trials examining the effect of supplementation of potassium apigenin on disease prevention have not been conducted, although there is considerable potential for apigenin to be developed as a radioprotective agent.

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).

203

Poster P.5.2.

Carnosic acid: a promising molecula for radiation protection

(1)Miguel Alcaraz; (4)Julián Castillo; (4)Obdulio Benavente García; (2)David Armero; (1)Miguel Alcaraz-Saura; (5)(*)Daniel Achel and

(3Vicente 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.

The genoprotective effect of carnosic acid against damage induced by ionizing radiation was compared with the effect of several antioxidant compounds by means of the micronucleus test for antimutagenic activity, in which the reduction in the frequency of micronuclei was evaluated in cytokinesis-blocked cells of human lymphocytes before and after 2 Gy of γ-radiation. Also, the radioprotective effect of the most effective compounds was then studied by a cell viability test (MTT) in the PNT2 (normal prostate) and B16F10 (melanoma) cell lines when they are administered before exposure to X ray doses (0Gy,4Gy,6Gy,8Gy and 10Gy).

The results point to the significant genoprotective capacity of the substances assayed before radiation: Rosmarinic acid

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(RO)=Carnosic acid (CA)=Diosmin (D)=Apigenin (AP)=δ-tocopherol (E)=Carnosol (Cl)=L_acid ascorbic (C)=Amifostine (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.

Cell survival obtained with 20 µM and 40 µM carnosic acid 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 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).

Carnosic acid showed the greatest pre-exposure protective effect, a good post-treatment protection capacity and no toxicity towards cells. For this reason, our results indicate that carnosic acid may be developed as a radioprotectant for humans against the potentially damaging effects of exposure to radiation.

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).

TOPICS: 9. Trace Elements and Human Health

205

Poster P.5.3.

Curcumin and copper binding curcumin induced apotosis in PC12 cells via

mitochodrial pathwaysNishimura R.1, Sato Y.1, Tanaka M.1, Miyajima M.1,

Hosokawa T.2, Kurasaki M.3, Saito T.1, 4

1Graduate School of Health Sciences, 2Institute for the Advancement of Higher Education, 3Faculty of Environmental Earth Sciences, 4Faculty of

Health Sciences, Hokkaido University, Sapporo, Japan

A hydrophobic polyphenol, curcumin has a variety of positive pharmacological, biological effects and protective effect against neurodegeneration in the central nervous system. The antioxidant activity of curcumin is enhanced by conjugated to copper and it is thought that there is some relationship to the curcumin and copper in the nervous system. However, the actions of curcumin with copper are complex and paradoxical in some cases.

In the present study, we investigated the effect of curcumin, copper (Cu) and copper binding curcumin (Cu-curcumin) on PC12 cell death. Firstly, cytotoxicity of PC12 cells exposed to curcumin, Cu and Cu-curcumin were measured by LDH assay. Treatment with 25µM, 50µM, 75µM and 100µM concentration

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of Cu for 24 hours had no effect on LDH leakage. LDH leakage induced by curcumin and Cu-curcumin significantly increased with dose-dependent manner. Although, LDH leakage induced by Cu-curcumin was significantly lower than curcumin-induced LDH leakage. Subsequently, to confirm whether curcumin and Cu-curcumin induced apoptosis or not, analyses of DNA fragmentation treated with curcumin and Cu-curcumin were carried out. Both curcumin and Cu-curcumin induced DNA fragmentation with dose-dependent manner. Interestingly, DNA fragmentation induced by Cu-curcumin was significantly higher than curcumin-induced DNA fragmentation. Moreover, to elucidate apoptosis pathway initiated by curcumin and Cu-curcumin, a release of cytochrome c from mitochondria was measured by western blot analysis. A Release of cytochrome c from mitochondria treated with 50μM Cu-curcumin was significantly higher than that in the control. Moreover, treatment with 50µM Cu-curcumin showed a notable increase accumulation of ubiquitinated proteins as compared with the control. These results suggested that curcumin and Cu-curcumin could induce apoptosis in PC12 cells via mitochondrial pathways and that Cu-curcumin enhanced the apoptosis process. Moreover, the Cu-curcumin-induced apoptosis is mediated through the inhibition of the ubiquitin-proteasome pathway.

207

Poster P.5.4.

Folate levels of vegetables commonly consumed in China

M. J. I. Shohag1*, Yanyan Wei1, Xiao-E Yang1,2* Jie Zhang1, Peter J. Stoffella2

1MOE Key Laboratory of Environmental Remediation and Ecosystem Health, College of Environmental and Resources Science, Zhejiang

University, Hangzhou, 310029, China; 2Indian River Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida,

Fort Pierce, FL34951-3138, USA. [email protected] or [email protected]

Folate deficiency increases the risk of chronic diseases, including neural tube defects (NTDs) in infants, megaloblastic anemia, cardiovascular disease and selected cancer in adults. China is the most NTDs prevalent area in the world. However, leafy vegetables are good source of folates but little is known about the folate content and vitamers among the vegetables commonly consumed by Chinese population. The purposes of this study were first to analyze most important commonly consumed vegetables that contribute to folate intake in the Chinese population and second to determine per capita folate intake from selected vegetables. We analyzed the folate content and vitamers forms in 26 most commonly consumed vegetables

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according to the consuming habit of Chinese people by using LC–UV/FLD. Mono-enzyme treatment for leafy green and fruit vegetables and di-enzyme treatment for some root vegetables with solid phase purification technique. Total folate content, expressed in folic acid equivalent (FAE) on fresh weight basis. 5-Methyltetrahydrofolate is the most predominant form in all vegetables analyzed. Total folate content in commonly consumed vegetables ranged from14.78 to 145.54 μg/100g FAE in edible portion with an average 61.99 μg/100g FAE. The highest folate content (145.54 μg/100g FAE) was found in Pak-choi. Total folate content in leafy vegetables, fruit vegetables and root vegetables were ranged from 17.22 to 145.54 μg/100g FAE, 18.14 to 86.04 μg/100g FAE and 14.78 to75.81 μg/100g FAE, respectively. Based on our folate analyses data and vegetables consumption data the dietary folate intake (Per- capita) of Chinese population from commonly consumed vegetables was estimated. The results indicated that considerable variations existed in folate content in different types of vegetables commonly consumed by Chinese population, and leafy vegetables are good source for folate than fruit and root vegetables that commonly consumed by Chinese population.

Keywords: Folate, Vegetables, genotypic difference, Per capita folate intake, China.

209

Poster P.5.5.

Evaluation of selenium and sulphur content in garlic (Allium sativum L.) as a possible source of organocompounds

with health benefitsOvesná J.1, Stavělíková H.1, Horníčková J.2, Velí ek J.2

1Crop Research Institute, 161 06 Prague 6, Czech Republic2Institute of Chemical Technology, 161 06 Prague 6, Czech Republic

Trace elements are important for human health as they regulate many processed and their concentrations in the tissues should be in a defined range. Cases of lower or high intake and their consequences have been reported. The essential trace mineral, selenium, is of fundamental important.as a constituent of selenoproteins. Selenium is known as an antioxidant and catalyst for the production of active thyroid hormone.

Several sources of organic selenium have been proposed, selenoproteins from garlic (Allium sativum L.) may be an important ones. Garlic is favourite vegetable being consumed world-wide having health benefits mainly due to organosulphuric compounds. Sulphurus may be substitute by selenium during

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organocompound sythesis. We aim to found ration of such substitution in various garlic varieties.

We have analysed content of sulphuric aminoacid in 135 garlic clones and registered cultivars and we found differences reflecting genetic background of the clones. Six selected clones with respect to morphotype and sulphuric aminoacid were treated by selenium salts and accumulation of selenium in leaves was investigated. Relationship between used concentration and accumulation was found. Results will be presented and discussed.

The work was supported by COST action 0905 and MEYS LD11066

211

Poster P.6.1.

Interaction of aluminum and boron in tea plants

R. Hajiboland, S. Bastani and S. Bahrami RadDepartment of Plant Sciences, 51666 University of Tabriz, Iran

There are interactions between Aluminum (Al) and boron (B) in plants at metabolism and structural levels because of similarities of the molecules and of the symptoms characteristics for Al toxic and B-deficient plants. Tea (Camellia sinensis (L.) O. Kuntze) plant is cultivated mainly on acid soils with high leaching losses of B and high Al availability. Tea is an Al accumulator species with growth stimulation in the presence of Al. We hypothesize that, responses of tea plants to Al supplementation will change under B deficiency, because of similar site of binding and change in the phenolics availability for Al chelation. Plants were grown from seeds and cultured for ten weeks in the nutrient solution pH 4.2 containing either low (<2.5 μM, –B) or adequate (46 μM, +B) B supply with Al supplementation at 300 μM (125 μM free Al3+ activity). Al supplementation in B-sufficient plants improved shoot and root growth. In B-deficient plants, Al-stimulated growth was observed particularly for root length. Al treated

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B-deficient plants had greater CO2 assimilation rate. Young leaves of Al treated B-deficient plants compared with B-sufficient ones have lower phenolic compounds in both cell wall bound (CB) and cytosolic (CS) fractions, while old leaves accumulated more CS phenolics and roots more CB phenolics. Al addition influenced B distribution between CB and soluble fractions. In turn, Al compartmentation was changed in B-deficient plants. Low B supply inhibited Al re-translocation from mature to young growing leaves. We suggest that a high CO2 assimilation rate, greater B root-shoot transport and increase in the cell wall bound B fraction are mechanisms for Al-mediated growth amelioration of B-deficient plants. Under these conditions, shoot Al allocated mainly to the old leaves and less Al was re-translocated into young leaves, where most Al was found in the cell wall-bound fraction.

213

Poster P.6.2.

Effect of selenium suplementation in nodulated alfalfa plants

Hajiboland R.1, Kontturi J.2, Rahmat S.1, Owusu A.2, Aliasgharzad N.3, Hartikainen H.4, Seppänen M.M.2

1Department of Agricultural Sciences, 00014 University of Helsinki, Finland2Department of Plant Sciences, 51666 University of Tabriz, Iran

3Department of Soil Sciences, 51666 University of Tabriz, Iran4Department of Food and Environmental Sciences, 00014 University of

Helsinki, Finland

Selenium is a beneficial element for higher plants. Mechanisms explaining the positive effect of Se on plants growth are not fully understood. Se supplementation activates the antioxidative defense capacity of plants and, thus, increases the resistance of plants against biotic and abiotic stresses. Under non-stress conditions, however, plant response to Se are likely attributable to other mechanisms. Alfalfa (Medicago sativa L.) is an important leguminous forage and fodder crop which is nodulated with Sinorhizobium meliloti. We investigated the effect of supplemental Se given as foliar spray or soil application on the nodulation of alfalfa plants. In the pot experiments, the growth, photosynthesis, nodulation, C:N ratio and nitrogenase

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activity were studied. Selenium was applied at 100 and 250 µg L-1 to the pots filled with perlite as growth medium, the plants were inoculated with Sinorhizobium meliloti and let to grow for two months. Hydrophonic cultures were used for studies on energy metabolism: sugar and starch accumulation and fructose 1,6-bisphosphatase activity. Results demonstrated that in both field and pot experiments the Se supplementation caused a slight but consistent increase of total biomass of plants (13%), fresh and dry weight (22%) of nodules, nodule/root fresh and dry weight ratio (13%) Net assimilation rate increased significantly (52%) in the Se-treated nodulated and non-nodulated alfalfa plants. However, neither nitrogenase activity per nodule weight nor C:N-ratio were affected by Se supplementation. On the other hand, sugar and starch content of leaves, stem and roots as well as F1,6-bisphosphatase activity in leaves increased significantly in the Se-treated plants. It seems probable that Se influences the C metabolism of plant via both increased stomatal conductance and activation of C-metabolizing enzymes.

215

Poster P.6.3.

Microelements accumulation in the mulberry trees grown in different

climatic conditionsLacatusu Anca-Rovena1, Lacatusu R.1; 2, Brailoiu Doina1,

Dumitru M.1

1 National Research & Development Institute for Soil Science, Agrochemistry and Environment Protection Bucharest, Romania; 2 “Al. Ioan. Cuza”

University Iassy, Romania

Two mulberry plantations in two different areas, conducted either in the classical or intensively cultivation system were investigated. The first plantation has been located in the western part of the Romanian Plain, on Chromic Luvisol, and the second in the central part of the Romanian Plain, on Luvic Phaeozem. The first soil has a medium texture, weakly acid reaction, and all other physical and chemical properties (aeration porosity, permeability, humus, total nitrogen, mobile phosphorus and potassium contents) at medium levels. The second soil has a fine texture, slightly acid-neutral reaction, low humus content, medium total nitrogen content, and high mobile forms of phosphorus and potassium supplies. In contrast to previous soils,

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a Limestone Arenosol located in the extreme east of the country, at Sulina belonging to the Danube Delta, was also studied. The main properties of this soil were coarse texture, alkaline reaction, medium CaCO3 content, high aeration porosity and permeability, high humus and low total nitrogen contents, low mobile forms of phosphorus and potassium content. On this soil, mulberry trees grew naturally. In the three investigated soils, the mobile microelements (Fe, Mn, Cu, Zn), soluble in CH3COONH4-EDTA solution at pH 7, supply levels were recorded medium values for the first two locations, and low values for the third. Analytical data of the metallic microelements (Fe, Mn, Cu, Zn) of active roots (Ǿ ≤ 1mm) and leaves showed normal content values in traditional culture conditions of the trees, and statistically increases up to 21 %, in intensive culture conditions. In contrast, levels of trace elements in roots and leaves of mulberry trees grown in the Sulina location were about 5-10% below the normal content. Among the microelements contents in leaves and roots, and mobile microelements contents in soil, the direct proportionally relationship, statistically assured for copper and zinc, were obtained.

Keywords: mulberry trees, metallic microelements, soil.

217

Poster P.6.4.

Necessity of biofortification of plants used as fodder or vegetable origin food

with selenium, in RomaniaLacatusu Radu1;2, Anca-Rovena Lacatusu1,

Mihaela Monica Stanciu-Burileanu1, Mihaela Lungu1

1 National Research & Development Institute for Soil Science, Agrochemistry and Environment Protection Bucharest, Romania; 2 “Al. Ioan. Cuza”

University Iassy, Romania

The experiments conducted into green house were followed the selenium enrichment of alfalfa, by the administration of selenium as potassium selenite solution (K2SeO3) into soil, on seed and on plant. Administration of selenium in soil, with doses from 1 to 10 mg / kg has contributed to increase the Se content of alfalfa plants up to 6.5 times. Foliar fertilization with solutions containing 4 mg Se in a volume of 100 ml water, increased the selenium content of the plant up to 14 times, and the application of selenium on seed by spraying it with solutions that contained between 1 and 5 mg Se / l helped to increase the amount of selenium in the plant up to 11 times. The experiments carried out on the field have revealed a tendency to increase the accumulation of selenium into the

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wheat grain, with up to 10% as a result of spraying the plants on 9-10 state according to Feeks scale with a solution containing 1 g sodium selenite (Na2SeO3) per hectare. The experimental data obtained show the necessity and possibility of bio-fortification with Se of the plants used as fodder and vegetable origin foods.

Keywords: selenite; alfalfa; wheat; biofortification.

219

Poster P.6.5.

Biofortification of cultures using phytochelatins

Juan Pedro Navarro AviñóUniversidad Jaume I, Av. de Vicent Sos Baynat, s/n 12071 Castelló de la

Plana

The phytochelatins (PCs) are polypeptides with a high content of cysteine, an amino acid capable of forming complexes with cations through the sulfhydryl group that has as radical. They are metal transport proteins responsible for the chelation of heavy metals in the cytoplasm of plant cells. Therefore, their role in tolerance and metal accumulation in plants is critical. This work shows the accumulation of oligoelements in wild (wt) and genetically modified (TAP) species of Nicotiana glauca including a phytochelatin synthase (PCS1) from wheat. It was observed that genetically modified plants show greater tolerance and accumulation of micronutrients present in soil. Specifically, the tolerance and accumulation data are significantly greater for the microelements B, Cu, Co, Ni and Zn. In addition, NgPCS1 enzyme homologous to TaPCS1, was cloned from Nicotiana glauca and overexpressed in yeast. For overexpression of NgPCS1, data

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similar to results of previous experiment with TaP plants were obtained. Following these results, and taking into account the deficiencies of micronutrients existing in human diets which are a Public Health problem especially in Third World Countries, this gene transfer to crop species could be an useful tool for further development of crop biofortification in those countries.

221

Poster P.6.6.

First report of low selenium concentrations in soil and bread-making

wheat grain in Guadalquivir Valley (Southern Spain)

López-Bellido F.J.1, Poblaciones M.J.2, Rodrigo S.2, López-Bellido L.3, Zhao F.J.4

1 Dpto. Producción Vegetal y Tecnología Agraria. University of Castilla-La Mancha. Spain.

2 Dpto. Dpto. Ingeniería del Medio Agronómico y Forestal. University of Extremadura. Spain.

3 Dpto. Ciencias y Recursos Agrícolas y Forestales. University of Córdoba. Spain.

4 Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, United Kingdom.

From Selenium (Se) is an essential microelement for humans with health benefits such as antioxidant, hormonal regulations of thyroid functions and anti-carcinogen effects. It is not essential for plants, which extract it from soil incidentally by the same route as for sulphate. Cereals are an important source of this element in the diet. In Spain there is scarce information on the intake of Se and its levels in the population, although the few known data suggest a low status in the population. The Se content is

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even less known in the agricultural soils and its extraction by the crops. Our aim was to make a preliminary study of the Se status in soil and wheat grain from samples belonging to the long-term experiment “Malagón”, located in a rainfed Vertisol in the Andalusian “Campiña”.

From the samples collection stored of this trial which started in 1986, samples of wheat grain and soil (0-30, 30-60 and 60-90 cm depths) were taken of two different years. Total Se concentrations were determined using aqua regia digestion for soil and nitric acid/hydrogen peroxide digestion for grain, followed ICP-MS analysis. Selenate and selenite in soil (bioavailable species) were determined by HPLC-IPC-MS.

Soil Se concentrations were similar in two years: 0.160 mg Se kg-1 (0-30 cm) and 0.172 mg Se kg-1 (0-90 cm). These concentrations can be considered “marginal” with regard to Se content. The Se bioavailable (selenate plus selenite) were always lower than 0.6 % of Se total and mainly in selenite specie. The concentrations of Se in wheat grain were also low, with slight differences between two years: 30 µg Se kg-1 (1996) and 44 μg Se kg-1 (2009). These preliminary results indicate low Se concentrations on Andalusian wheat, explaining in part the low levels of Se in blood plasma of its population. Further work is needed to know in depth the Se wheat concentrations in this region and the factors influencing uptake from soil.

223

Poster P.7.1.

Trace elements in black, green, red and white tea leaves

Beatriz Pérez Marrero, Gara Luis, Carmen Rubio, Cintia Hernández Sánchez, Ángel Gutiérrez, Arturo Hardisson.Área de Toxicología de la Universidad de La Laguna, S/C de Tenerife,

Canarias.

Tea is the second most consumed beverage in the world. The main varieties consumed are: white tea (young shoots), red tea (semi-fermented), black tea (fermented) and green tea (unfermented). The aim of this study is to determine the content of Co, Cr, Cu, Fe, Mn, Ni and Zn in 4 varieties of tea. For this,80 samples of 4 varieties of tea (20 samples of each variety) were analyzed. Co, Cr, Cu, Fe, Mn, Ni and Zn contents were determined by Flame Atomic Absorption Spectrometry (AAS). The obtained results show Co mean levels: 0.70 ± 0.10; 0.46 ± 0.20; 0.44 ± 0.09 y 0.45 ± 0.16 mg/kg for white, green, black and red tea, respectively.

Cr mean contents: 1.12 ± 0.38; 0.26 ± 0.08; 0.38 ± 0.15 y 2.56 ± 0.54 mg/kg for white, green, black and red tea, respectively.

Cu mean concentrations: 5.46 ± 1.32; 8.40 ± 4.50; 5.64 ± 1.19 y 7.76 ± 1.33 mg/kg for white, green, black and red tea, respectively.

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Fe mean values: 90.00 ± 28.85; 65.66 ± 26.77; 33.29 ± 13.72 y 227.43 ± 68.39 mg/kg for white, green, black and red tea, respectively.

White, green, black and red tea showed 142.33 ± 103.22; 38.13 ± 19.79; 58.03 ± 38.53 y 100.00 ± 41.85 mg Mn/kg, respectively.

The observed Ni mean contents were 3.38 ± 0.86 mg/kg for white tea; 2.31 ± 0.54 mg/kg for green tea; 2.82 ± 0.53 mg/kg for black tea and 3.87 ± 0.48 mg/kg for red tea.

White, green, black and red tea presented 7.35 ± 1.60; 5.16 ± 1.11; 5.04 ± 0.81 y 7.20 ± 1.06 mg Zn/kg, respectively. Finally, we can conclude that while red tea shows the higher Cr, Fe and Ni levels, white tea is the richest in Mn, Zn and Co and the poorest in Cu. Green tea presents the highest higher Cu concentrations and, at the same time, is the poorest variety in Cr, Mn and Ni. The lowest Co, Fe and Zn levels were detected in black tea.

225

Forensic analysis of human teeth by laser-induced breakdown spectroscopy

F.J. Fortes1, M.D. Pérez-Cárceles2, A. Sibón3, A. Luna2, J.J. Laserna1

1 Department of Analytical Chemistry, Faculty of Sciences, University of Malaga

Campus de Teatinos s/n, 29071 Malaga, Spain2 Department of Legal and Forensic Medicine, Faculty of Medicine,

University of MurciaCampus de Espinardo, 30100, Murcia, Spain

3 Institute of Forensic Medicine. Cádiz. Murcia

The recovery of a corpse from the water raises always multiple and varied questions. The need for elemental analysis of biological matrices such as bone and teeth for discrimination purposes has emerged within the forensic laboratory. Bones and teeth are commonly found in crime scenes and the analysis of the trace evident content could contribute additional information for use in forensic investigations. In this sense, teeth are found to maintain much of the biological signature from the living phase over a long time, revealing for example the absorption of contaminants from the surrounding environment.

Poster P.7.2.

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Nowadays, modern analytical techniques such as SEM-EDX, XRF, ICP-MS and GC-MS are normally used as routine in laboratory analysis with high precision and accuracy in the results. In the last few years, laser-induced breakdown spectroscopy (LIBS) has emerged as a promising method in the examination and determination of trace evidence in forensic analysis. The particular advantages of LIBS in terms of no sample preparation, fast analytical response, and potential for field portability make this method attractive to forensic science.

In this work, we report on the application of LIBS to the analysis of important minerals and potentially toxic elements within the teeth matrix. For this purpose, a set of human teeth has been examinated by using a conventional LIBS configuration. A Q-switched Nd:YAG laser operating at 1064 nm was employed to generated plasmas at the sample surface. Chemical evidences detected by LIBS may offer crucial information on the cause of death. In this sense, special distribution of the chemical evidences in the human teeth will be presented and discussed.

227

Cu and Zn in 2009 wines from 5 Canary Islands Designation of Origin

Naoual Ben-Charki1,2, Gara Luis1, Carmen Rubio1, Juan Manuel Méndez2, Ángel Gutiérrez1, Arturo Hardisson1

1 Área de Toxicología de la Univerisidad de La Laguna, S/C de Tenerife, Canarias, España.

2 Instituto Canario de Calidad Agroalimentaria. Santa Cruz de Tenerife.

The 1980´s marked the re-emergence of the Archipelagos wines which now have eight Designations of Origin: Tacoronte-Acentejo, Ycoden-Daute-Isora, Abona, Valle de Güimar and Valle de la Orotava, on the Islands of Tenerife as well as Lanzarote, El Hierro and La Palma. The aim of this study is Cu and Zn determination in red and white wines from 5 different Designations of Origin (DO) in the Canary Islands (La Palma, La Gomera, Tacoronte-Acentejo, Güimar and Abona). To do this study, 188 samples (105 red wine samples and 83 white wine samples) of 2009 bottled wines were analyzed. Cu and Zn contents were determined by Flame Atomic Absorption Spectrometry (AAS). No previous sample treatment was required. Finally, Cu mean levels and standard deviations were 0.2014 ± 0.27998 and 0.1917 ± 0.40272 ppm for red and white wines,

Poster P.7.3.

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respectively. Zn mean contents and standard deviations were 0.4394 ± 0.42343 y 0.5783 ± 0.60449 ppm for red and white wines, respectively. According to the different designations of origin, the mean Cu and Zn concentrations and standard deviations were:

Cu mean levels according to the different DO were La Palma: 0.1719 ± 0.34512 ppm; La Gomera: 0.1962 ± 0.14935 ppm; Tacoronte-Acentejo: 0.3075 ± 0.45968 ppm; Güimar: 0.1572 ± 0.28281 ppm and Abona: 0.1145 ± 0.16230 ppm.

Zn mean values according to the different DO were La Palma: 0.633 ± 0.24954 ppm; La Gomera: 1.0486 ± 0.70056 ppm; Tacoronte-Acentejo: 0.6975 ± 0.59739 ppm; Güimar: 0.4008 ± 0.22418 ppm and Abona: 0.2918 ± 0.27152 ppm. Finally we can conclude that mean levels of Zn in wine samples of different DO are higher than average concentrations of Cu. Wines from La Palma island show lower Cu and Zn levels than wines from La Gomera and Tenerife islands.

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Mineral composition of wild and cultured Sea bass (Dicentrarchus labrax, L.)

M. Santaella, C. Martínez Graciá y Mª J. Periago Department of Food Technology, Nutrition and Food Science. Area of

Nutrition and Food Science. Faculty of Veterinary Science. Universidad de Murcia. Campus de Espinardo 30100, Murcia, Spain.

Reference Author: Marina Santaella. Email: [email protected].

Sea bass (Dicentrarchus labrax, L) is a fish specie of high commercial value with important market prospects. Its high quality coupled with growing demand and a limited supply of fishing, have stimulated interest in its cultivation, which has experienced a considerable increase in recent years. The desirable aroma and quality attributes of the wild specimens have lead to state this type of fish as the standard of quality. Aquatic organisms absorb minerals from the diet and surrounding water and deposit them in their skeletal tissues and organs. Fish muscle serves as a good source of essential minerals. The composition of commercial feed used for cultured fish also influences the mineral composition of the fish. Wide variations have been observed in the reported values of mineral concentrations in the same species of fish depending of the type of origin.

Poster P.7.4.

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We analyzed K, P, Na, Ca, Mg, Fe and Zn concentrations in flesh from 10 specimens of wild (Mediterraneal cost) or cultivated (Instituto Oceanográfico de Mazarrón, Murcia) Sea bass. After dry ashing, samples were dissolved in hot concentrated hydrochloric and nitric acid, filling up with deionised water to a suitable volume. The mineral analysis was performed by an atomic absorption spectrophotometer Perkin Elmer Model 3100. To prevent interferences, samples were treated with lanthanum chloride at 0.1%. The P was analyzed using the colorimetric method described by AOAC (1990) (Method 965.17).

The flesh of wild Sea bass showed higher concentrations of K, Na, Fe and Zn, appearing statistically significant at p <0.05. Numerous studies have shown that the concentration of minerals is influenced by various factors such as season, the chemistry and salinity of the water, pollutants, height, age, sex, stage of sexual maturity and the relationship between red and white muscle Thus the higher content of Fe and Zn of wild sea bass (6.45 ± 2.15 mg/100 g and 0.0 ± 0.07 mg/100 g, respectively), may be due to a predominance of red fibers with respect to the white, as red muscle is characterized by a higher content of trace elements. Furthermore, the iron of muscle myoglobin is in the form of heme-iron, with higher bioavailability than nonheme-iron forms mainly found in foods of vegetable origin.

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Use of ultrasound-assisted dispersive liquid-liquid microextraction followed

by electrothermal atomic absorption spectrometry for the speciation of

chromium released from toysI. López-García, M. Briceño,

N. Campillo, and M. Hernández-CórdobaDepartment of Analytical Chemistry, Faculty of Chemistry, University of

Murcia, E-30100, Murcia, Spain.

The toxicological and biological characteristics of many elements depend on their chemical forms. Thus, it is well known that Cr(III) appears to be one of the essential elements for the proper functioning of living organisms, while water soluble Cr(VI) is toxic to humans and classified as carcinogenic. Besides obvious matrices to be controlled such as waters, a case of particular relevance is that of the Cr(VI) present in toys. Children may be exposed to heavy metals when playing with these objects. In addition to the residues in fingers and hands, there is a risk if the toys they are playing with enter the mouth. The convenience of

Poster P.7.5.

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sensitive analytical methods based in techniques easily available in all the laboratories for the purpose of measure the low amounts of chromium that can be released is clear.

This communication reports a new procedure for the purpose, with the advantage of differentiate between the two above mentioned oxidation states. The procedure is based in the use of a recently introduced way of preconcen-tration (dispersive liquid-liquid microextraction, DLLME). The sample is extracted with a few microliters of tri-n-butyl phosphate (TBP) in HCl medium, the extraction being almost instantaneous using ultrasounds to obtain a suitable dispersion. An aliquot of the organic liquid is then introduced into the electrothermal atomizer of an atomic absorption spectrometer, and the concentration of Cr(VI) measured. Since only the hexavalent form is extracted, speciation is possible by means of a second extraction in the presence of a suitable oxidant. This simple, easy-to-perform and environmentally friendly procedure (the volume of organic reagent used is very low) allows extremely low amounts of chromium to be measured. The procedure has been applied to the determination and speciation of chromium in several toys. For this, the samples were previously treated as indicated in the European method EN 71. Data for several toys are given.

233

Poster P.8.1.

Natural genetic variation controlling zinc use efficiency in Arabidopsis

1Ana Carolina A. L. Campos, 1Ariadne R. Henriques, 1Charles, N. Moreira, 2Ana G. L. Assunção, and 1Mark G. M. Aarts

1 Wageningen University, Laboratory of Genetics. P.O. Box 309, 6700AH, Wageningen, The Netherlands

2 University of Copenhagen, Department of Plant Biology, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark

Zinc is a micronutrient required as a cofactor for many enzymes involved in important biochemical pathways. Leaf Zn concentrations below 15 to 20 μg per g dry mass lead to Zn deficiency symptoms, which are associated with early senescence and chlorotic leaves and often result in stunted growth, thus causing yield losses of 40% or more for crops in Zn deficient soils. As a consequence of coping with low zinc availability some plant genotypes developed the ability to grow and yield under Zn limiting conditions. This process is named Zn Use Efficiency (ZUE). There is not much information about the genes controlling ZUE in plants. This study aimed to determine the natural variation related to ZUE present in the HapMap Arabidopsis collection, comprised of 360 accessions collect in different

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regions of the world. Also, the Zn translocation in mutants with a differential response of the ZIP4 gene to Zn deficiency conditions was characterized. We could observed significant natural genetic variation in the set of 20 Arabidopsis accessions grown hydroponically under low and normal Zn and later on when the whole HapMap collection was grown in the same conditions. These results indicate that we have good prospects to identify QTLs by genome-wide association mapping. When two accessions contrasting in the ZUE (efficient versus non-efficient) were grown in agar plates their root growth was strongly affect by the Zn deficiency. In addition, the zinc content of shoot and root tissues of the mutants was determined under optimal and low Zn supply. At low Zn M31 showed an aberrant root to shoot Zn allocation, with more Zn in roots than the WT. At normal Zn M22 and M60 showed elevated shoot Zn concentrations compared to the WT.

Key-words: Arabidopsis HapMap collection, mineral nutrition, zinc deficiency.

Supported by: CBSG (Centre for BioSystems Genomics).

235

Poster P.8.2.

Effects of heavy metals on the detoxification system of Typha latifolia

Peter Schröder, Lyudmila Lyubenova Helmholtz Zentrum München German Research Center for Environmental

Health, Department Microbe-Plant Interactions, Neuherberg, Germany.

Heavy metals are ubiquitous in the environment and their presence in a given ecosystem can lead to accumulation in the food chain with negative effects for human health. Even minute heavy metal concentrations can provoke alterations in cellular processes and structures in plants. It is well documented that higher metal concentrations cause the generation of ROS. If ROS support is not controlled oxidative chain reactions will start. Although oxidation can be withstood to a certain extent by proteins, nucleic acids, lipids, plants usually respond to this stress by increasing activities of antioxidant enzymes. The antioxidant defence system includes enzymes like superoxide dismutase, catalase, ascorbate peroxidase, glutathione peroxidase, and glutathione S-transferases.

The present study aimed at quantifying the effects of sublethal concentrations of heavy metals on a typical plant used in

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phytoremediation, Typha latifolia. As waste water treatment plants usually exhibit intermittent high concentrations of heavy metals, the detoxification capacity of T. latifolia treated with heavy metals during 72 h was investigated. Furthermore, the defence mechanisms in cattail were explored with respect to enzymatic steps providing efficient tolerance against organic xenobiotics.

Upon treatment with Cd and As T. latifolia showed induced catalase, monodehydroascorbate reductase and ascorbate peroxidase activities in leaves but strong inhibition in rhizomes. Peroxidase activity in leaves of the same plants was inhibited whereas linear increase was detected after Cd treatment in rhizomes.

Glutathione S-transferase measurements resulted in identical effects of the trace elements on the substrates CDNB, DCNB, NBC, NBoC, fluorodifen. When GST was assayed with the model substrate DCNB, a different pattern of activity was observed, with strongly increasing activities at increasing HM concentrations.

Consequently, to improve the success rates, future phytoremediation plans need to preselect plant species with high antioxidative enzyme activities and an alert GST pattern capable of detoxifying an array of organic xenobiotics.

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Poster P.8.3.

Use of alkaline materials in humic podzols: a way to reduce cd in vegetables

and cerealsMench Michel1, Verdal Marie-Noëlle,

Solda Paul, Lecuyer Isabelle1UMR BIOGECO INRA 1202, Ecology of Communities, University of

Bordeaux 1, Bât B8, RdC Est, avenue des Facultés, F-33405 Talence, France. [email protected].

Keywords: cabbage, cadmium, celeriac, food safety, French bean, leek, lettuce, maize, potatoes, soil pH, turnip, wheat.

The contributions of vegetables plus fruits and cereals to the daily dietary intake of cadmium of French adults (> 15 year-old) average 1.4 and 0.3 μg Cd/d respectively (Leblanc et al, 2005). This corresponds to 62 % of the daily dietary intake of Cd for a French adult. For high consumers (95th percentile), the contributions of vegetables plus fruits and cereals can peak at 4.9 and 1.1 μg Cd/d. The Cd concentration of some vegetables with a Cd accumulator phenotype such as lettuce may exceed the EU MPC (maximum permitted concentration) in acid humic podzol from Southwest France. Liming is a potential agricultural

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practice to reduce labile Cd pool in the soil and to minimise the root exposure and uptake. Therefore, three alkaline materials, i.e. calcium carbonate (CaCO3, 3.8 and 15.9 t/ha), coal bottom ash (CBA, 7.8 and 30.4 t/ha) and coal fly ash (CFA, 7.1 and 17.1 t/ha) were incorporated into a humic podzol in a field experiment which increased soil pH in year 1.5 from 4.6 to 5.9-6.7, 5.3-6.1, and 5.6-6.3, respectively. The CaCO3, CBA, and CFA inputs of Cd were <3.8-<16, 0.8-3, and 2.8-6.8 g/ha. Winter wheat, maize, cabbage, celeriac, French bean, leek, lettuce, potatoes, turnip and carrots were cultivated and harvest at market stage. All plant parts were carefully washed 3 times with distilled water, cut with ceramic scissors, weighed (fresh weight, FW), oven dried (45°C) till constant dry weight (DW), and ground (Restch). Potato tubers, roots of turnip and carrots were pealed prior to be oven-dried. Weighed aliquots of plant materials (0.5 g DW) were wet-digested with microwaves (MarsXpress, CEM) with HNO3/H2O2. Element concentrations in digests were determined with ICP-AES (Varian liberty 200) and GFAAS (Varian Zeeman effect). Quality scheme included blank reagents and certified reference materials in all sample batches. Certified reference values were recovered (>95%). Plant samples were analysed in triplicates (all values expressed in mg/kg DW). Cadmium concentrations in lettuce (1.6) from the untreated soil exceeded the EU MPC value. Other plant samples were below the Cd-EU MPC value. In untreated soil, Cd concentrations in edible plant parts ranged in the following order: corn grain < cabbage, French bean < wheat grain < potato tubers < turnip < leek, celeriac < lettuce. Liming with CaCO3 decreased Cd concentrations in lettuce (-50%), turnip, peeled potato tubers, and leek. Shoot Cd concentration in lettuce negatively correlated with soil pH. Both CBA treatments did not decrease lettuce Cd concentrations, despite increase in soil pH. Lettuce Cd concentrations were higher in CFA-treated

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plots than in CaCO3-limed-plots at similar soil pH, and increased with the CFA application rate. Liming with CaCO3 up to 6.7 had no effect on Cd concentration in cabbage, French beans, and celeriac. In CaCO3 limed-plots, grain Cd of winter wheat significantly decreased from 0.08 to 0.06. Grain Pb concentration remained in all plots below 0.09. At 7.8 t CBA/ha, the turnip Cd concentration peaked at 0.4 mg Cd/kg DW. Accounting for the vegetable amounts ingested per year and adult, liming with CaCO3 allowed diminishing dietary intake from 2.6 to 1.3 mg Cd/yr/adult. Dietary intakes in the case of CBA and CFA-limed soils, i.e. 2.6-2.5 and 1.7-2.1 mg Cd/yr/adult, would be higher than in CaCO3-limed plots.

241

Poster P.8.4.

Preventing toxic element penetration into plants and fruits by using grafted

plants Menahem Edelstein1, Zvi Plaut2, Meni Ben-Hur2

Department of Vegetable Crops & Plant Genetics, Agricultural Research Organization, 1Newe Ya‘ar Research Center, P. O. Box 1021,

Ramat Yishay 30-095, Israel2Institute of Soils Water and Environmental Sciences, Agricultural Research

Organization, Volcani Center, P. O. Box. 6, Bet Dagan 50-250, Israel

The use of marginal water for irrigation is increasing in arid and semiarid regions. Saline waters can contain high concentrations of salts and toxic microelements. Long-term use of these waters for irrigation could increase the accumulation and concentrations of microelements and saline elements (Na, and Cl) in the soil. Relatively high concentrations of Na+, Cl-, and microelements in the soil solution could be toxic to plants and to human beings. Absorption of these elements by the plants could affect their growth and yield, and increase the possibility of entry of contaminants into the human food supply chain. The effects of grafting on uptake and distribution of toxic microelements and Na within the plant were determined in two experiments, one

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in the field using non-grafted melon (Cucumis melo L. ‘Arava’) plants and melon plants grafted on pumpkin (Cucurbita maxima Duchesne × Cucurbita moschata Duchesne ‘TZ-148’), and one in the greenhouse using six combinations of melon and pumpkin plants: non-grafted, self-grafted, melons grafted on pumpkins and pumpkins grafted on melons. In the field experiment, non-grafted melon plants accumulated in their shoots and fruits more Na and Cl than those grafted on pumpkins. Moreover, the concentrations of the microelements B, Zn, Sr, Mn, Cu, Ti, Cr, Ni, and Cd in melon fruits from melon plants grafted onto pumpkin plants were lower than from non-grafted melon plants. In the greenhouse experiment, boron concentrations in the exudates from plants with melon root systems were higher than plants with pumpkin root systems. Grafting appears to be potentially useful for reducing the accumulation of toxic elements in fruits and thus in food-supply chains. On the other hand it is also possible to find rootstocks with ability to increase the absorption of trace elements.

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Poster P.8.5.

Understanding basis of magnesium use efficiency in plants

Hermans C. and Verbruggen N.Lab of Plant Physiology and Molecular Genetics, Université Libre de

Bruxelles, Belgium [email protected]

Magnesium has become a priority metal because in addition to be an essential element in plant cell biology, Mg is the 4th most common cation in the human body and half of its dietary intake is from plant origin, mainly from chlorophyll in green vegetables. Mg deficiency is a widespread problem, affecting productivity and quality in agriculture [1]. Hypomagnesaemia in the human body is recognized as a global clinical problem.

Our overall goal is to gain insight into the genetic determinants of Mg homeostasis in plants, a domain relatively unexplored up to date [2]. Our research lines are the exploitation of ionomic variation and the analysis of transcriptome changes associated with Mg availability in Arabidopsis, with a possible transfer to Brassica crops.

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First, we studied the natural variation in accessions as a source of biodiversity to find new genes and new alleles controlling Mg content in tissues. We observed an 60% difference variation in the Mg concentration of the roots and of 50% in the shoots between the most extreme accessions of the Nordborg Arabidopsis collections. Currently QTL analysis between couples of contrasted couples allows the identification of an interesting locus.

Second, we recently published a thorough description of the early transcriptomic responses to Mg deprivation [3,4]. We observed alterations of the circadian clock after Mg depletion, the triggering of the ethylene biosynthetic pathway and the photoprotection of the photosynthetic apparatus

Through the understanding of Mg homeostasis mechanisms, this work may provide new tools to biofortify Mg concentration and to improve the tolerance to Mg deficiency in Brassica crops. A perspective of this work is to help in the identification of molecular markers for breeding purpose.

References

[1] Bennett WF (1997) Nutrients Deficiencies & Toxicities in Crop Plants. APS Press, The American Phythopathological Society.

[2] Hermans C. and Verbruggen N. (2008) Dietary Magnesium: New Research. ISBN 978-1-60692-109-8, pg. 159-175.

[3] Hermans C. et al. (2010) New Phytol. 187, 119-131.

[4] Hermans C. et al. (2010) New Phytol. 187, 132-144.

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Poster P.8.6.

Current level of lead and cadmium in edible and pasture plants growing in areas with elevated metal content and methods for diminishing metal

concentration in plantsJadwiga Gzyl, Marta Pogrzeba, Jacek Krzyżak

Institute for Ecology of Industrial Areas, 6 Kossutha St., 40-844 Katowice, Poland, [email protected]

Some European regions with elevated levels of Pb and Cd in soil are still using their areas for agricultural production. As a consequence, bodies responsible for food safety have to struggle with the exceeded content of these metals in food and feed.

In this paper concentrations of Pb and Cd in vegetables, potatoes, cereals and pasture plants in such areas will be analyzed. Evaluation of Pb and Cd content in plants in the context of European Union permissible standards will also be presented. A review of the collected data shows that a significant part of the plant samples exceeds EU standards.

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The above-mentioned problems should be solved by applying specific methods for diminishing metal bioavailability in plants, i.e.:

— soil liming as a method of pH rising,

— improving soil quality by adding organic matter (i.e. lignite).

There are also methods for protection from soil contamination, such as:

— control of metal content in phosphorus fertilizers,

— control of metal content in sewage sludge used as organic matter.

Preliminary results for crop quality improvement methods will be presented.

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