© azti-tecnalia final conference safefoodera meeting copenhagen, 7th june 2011 alejandro barranco...
TRANSCRIPT
© AZTI-Tecnalia
Final Conference Safefoodera meetingCopenhagen, 7th June 2011
Alejandro BarrancoAZTI-Tecnalia, Food Research Division, Spain
e-mail: [email protected]
Bioavailability and risk assessment of polycyclic aromatic hydrocarbons (PAHs) and toxic elements (As, Cd, Hg and Pb) in
processed meat and seafood products
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IPIMAR (Portugal) Portuguese Governmental Research Institute for
Fisheries and Sea Contaminants in seafood and risk analysis
Riskfoodcont Partners
INIA (Portugal) National Institute of Agricultural Research Contaminants in meat products
University of Maribor (Slovenia) Department of Microbiology, Biochemistry, Molecular
Biology and Biotechnology Cell lines
AZTI-Tecnalia (Basque Country)Detection system areaZebrafish and biosensors
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Objectives and structureWhy this project?Objectives and workplanMethodology
Main resultsSeafood Edible crab Black scabbard fish
Meat products Smoked meat products
Conclusions
Outline
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Human exposure to contaminants
They are in the environment (air, soil…)
Contact with contaminated materials
Residues in food
Why RISKFOODCONT?
Heavy metalsPAHs
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Heavy metals
Why RISKFOODCONT?
Number of notifications in UE. Source: RASFF anual report 2009
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Heavy metals
Why RISKFOODCONT?
Number of notifications in UE. Source: RASFF anual report 2009
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Heavy metals
Why RISKFOODCONT?
Mercury: Fish and seafoodMRL: 0.1-1 mg/kg
Cadmium: Meat, fish, cerealsMRL: 0.05-3 mg/kg
Lead: Milk, meat, fish, cereals
MRL: 0.02-1,5 mg/kg
Tin: Canned food
MRL: 20-200 mg/kg
Organo metallic compounds
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Polycyclic aromatic hydrocarbons
Why RISKFOODCONT?
Ubiquitous contaminants
Source: Combustion of organic materials with low amount of
oxigen
>90% of exposure occurs via food residues
MRL: 1-10 ug/kg benzo[a]pyrene (edible oils and fats,
smoked fish and meat, fresh fish and seafood)
Very lipophilic compounds
Toxic, carcinogenic and mutagenic by long term exposure
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Polycyclic aromatic hydrocarbons
Why RISKFOODCONT?
Light PAHs
Heavy PAHs
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Objectives
Implement innovative research tools (e.g. pig and human
cell lines, zebrafish embryo, toxicogenomic) to study the
bioavailability and risk assessment of PAHs and heavy metals in
processed food products, using smoked meat sausage and
cooked edible crab and black scabbard fish as models
Identify specific toxicity biomarkers through the analysis of
the toxicogenomic response to contaminant exposure
Develop rapid methods able to detect small amounts of
toxicants in foodstuff even if masked by the coexistence of other
compounds or specific food matrices
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Workplan
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Methodology
Toxicological studies
Cell lines
– 2 types of pig cell lines (PSI y CLAB)
– Human cells (H4)
Microporous membraneMicroporous membrane
Apical compartmentApical compartment
Basal compartmentBasal compartmentPPolarised olarised
intestinal cell intestinal cell linelines:s:
• CLAb and PSICLAb and PSI• H4H4
Monocyte/macrophageMonocyte/macrophage cell cell lines:lines:•Pom Pom •TLTTLT
Methodology
Toxicological studies. Zebrafish
• Asiatic sweet water fish of ≈ 5 cm
• Breed all year round.
• Time of generation: 3-5 months.
• Lay up to 300 eggs. Externally fertilized.
• Share many characteristics with vertebrates
• Its genome has been completely sequenced
• Embryos and larvae are transparent
• Not considered animals
Zebrafish embryos and larvae
5dpf5hpf
Alternative test
alternative tests
Methodology
Toxicological studies. Zebrafish
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Larvae Expresion profile
48 h exposure DNA chip
MicroarrayValidation of biomarkers
qRT-PCR
Biomarker genes/proteins
ENDPOINTS PARAMETER ANALYSIS
Morphology Malformation
Pigmentation Microscopic analysisDevelopment Delay
Death
Molecular Differential expression analysis toxicity marker genes
qRT-PCR
Methodology
Bioavailability and culinary treatments
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Protocol to simulate the things occuring at:-mouth-stomach-intestine
Raw (control)SteamedGrilledFried
60 specimens60 specimens
Raw (control)SteamedBoiled
Black scabbard fish muscleBlack scabbard fish muscle
Methodology
Risk assesment
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– Surveys about habits of consumption
– Residues of target contaminants in food samples
– Bioavailability and tosic effects
– Several scenarios were proposed:
150 g de fish/meal; 70 Kg adult
PTWI (Hg): 5 µg Hg/Kg/semana
– Software: @RISK
Biosensors
– Identification of suitable biomarkers
– Development of inmunoassays
Main reults
Toxic effects
– Zebrafish. Metals
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Low concentration: 0.14 ppm CdCl2 + 9 ppb HgMetCl
Medium concentration: 0.5 ppm CdCl2 + 50 ppb HgMetCl
High concentration: 1.4 ppm CdCl2 + 100 ppb HgMetCl
Main reults
Toxic effects
– Zebrafish. Metals
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0.14 ppm CdCl2 + 9 ppb HgMetCl
Main reults
Toxic effects
– Zebrafish. PAHs
– No mortality was observed
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1ppb
10ppb
Main reults
Bioaccesibility and culinary treatments
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Hg
(m
g k
g -1
)
Limit = 1.0 mg kg-1
Black scabbard fish
More Hg in grilled samples due to the loss of water
Cd
(m
g k
g -1
)
Limit = 0.50 mg kg-
1
Edible crab (Hepatopancreas)
More Hg in grilled samples due to the loss of water
Main reults
Bioaccesibility and culinary treatments
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% M
erc
ury
Hg is more accesible at the intestine and in the case of raw fish
Gastric step: 1.3 – 3.3% Hg
Gastrointestinal step : 5.8 – 14.5% Hg
Main reults
Bioaccesibility and culinary treatments
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Cd is more accesible at the intestine and in the case of raw fish
Gastric step: 45 - 72% Cd
Gastrointestinal step : 83 – 90% Hg
Main reults
Bioavailability
– Cell lines. Methylmercury
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Very low amount (<4%) of Methylmercury goes through the cell lines Transport lower with digested samples
Main reults
Bioavailability
– Cell lines. Methylmercury
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Higher bioavailabillity with real food samples
Main reults
Bioavailability
– Cell lines. Cadmium
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Up to 30% of Cadmium goes through cell lines
More transport with digested solutions
Main reults
Bioavailability
– Cell lines. Cadmium
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All samples did not show the same behaviour
Main reults
Surveys
– Portugal. 800-2000 answers
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Edible crab
97 % consume hepatopancreas
Consumption of smoked meat products
Main reults
Residues
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Main reults
Risk assessment
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Black scabbard fish
PTWI = 5 g Hg/kg/week
. Fryed: 1:12,000,000 1:14,000,000
. Grilled: 1:3,000,000 1:1,800,000
Adults Children
Edible crab
Adults and boiledHepatopancreas– 1:30Whole– 1:80Muscle – 1:641,000,000
TWI = 2.5 g Cd/kg body weight
Despite the Hg content, low risk to exceed PTWI
The consumption of hepatopancreas is not recomended
Main reults
Biosensors• Mt and HSP70 genes in the case of metals
• A strong induction of cytochrome P450 family C1 (CYP1A1 gene) has been observed in the case of PAHs
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Design of an ELISA kit for the simultaneous of target biomarkers
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Conclusions
Innovative and more realistic tools have been applied to evaluate
risk assessment of single and multiple contaminants in food
samples.
Zebrafish is a good model for investigating the effect of food
contaminants as a preliminary screening. Intermediate toxocity
assay between in vitro and in vivo tests
Excellent tool for identifying toxicity biomarker genes
It is important to know the mechanism of toxicity through our
digestive system. In this sense, cell lines are an useful tool.
Contribute to set new limits for PAHs and heavy metals in food
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Conclusions
Culinary treatments play an important role. In the case of mercury
bioavailability was higher in raw fish
Despite the relatively high levels of Hg in cooked black scabbard
fish, the risks of exceeding PTWI are extremely reduced
When consuming edible crab, consumers should reduce the brown
meat portion to the lowest level and prefer the muscle
Risk benefit analysis
Dissemination
- 3 national conferences /workshops
- Articles (14) and congresses (9)
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www.azti.es
Thank you for your attention!!!
www.azti.es
Riskfoodcont team