© AZTI-Tecnalia Final Conference Safefoodera meeting Copenhagen, 7th June 2011 Alejandro Barranco AZTI-Tecnalia, Food Research Division, Spain e-mail:

© 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

© AZTI-Tecnalia

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