Heterocyclic Aromatic Amines in Muscle Foods - Repositório Aberto

176 Pages·2012·5.06 MB·English

Heterocyclic Aromatic Amines in Muscle Foods -
Relevance, Occurrence and Mitigation

Olga Maria da Silva Viegas

Porto
Março 2012

Dissertation Thesis

Heterocyclic Aromatic Amines in Muscle Foods -
Relevance, Occurrence and Mitigation

Dissertation presented to Faculty of Nutrition and Food Science to obtain the degree of
Doctor of Philosophy (PhD) in Food Consumption and Nutrition Sciences

Supervisior: Professor Doctor Olívia Maria de Castro Pinho
Co-supervisor: Professor Doctor Isabel Maria Pinto Leite Viegas Oliveira Ferreira

Olga Maria da Silva Viegas
Porto, 2012

Olga Viegas is grateful to Fundação para a Ciência e a Tecnologia for a PhD grant
(SFRH/BD/42122/2007) financed by POPH-QREN and subsidized by ESF and MCTES
The studies presented here were done at REQUIMTE Laboratory of Bromatology and Hidrology of
Faculty of Pharmacy of the University of Porto, at Faculty of Nutrition and Food Science of
University of Porto and at the Department of Genetic Toxicology and Cancer Biology of National
Institute of Biology, Slovenia.
Acknowledgments

I would like to formally express my sincere gratitude to...

… my supervisors Professor Doctor Olívia Pinho and Professor Doctor Isabel Ferreira. I feel
honored for the opportunity to do my thesis. I am very grateful for the strict scientific guidance that
was, without any doubt, the key to the effective progression and success of my research work. All
your advices, corrections, constant encouragements, patience and optimism were essentials for
complete this work.
… to Professor Doctor Metka Filipič, for the great supervision in the Department of Genetic
Toxicology and Cancer Biology – National Institute of Biology, Slovenia. Thank you for sharing
your knowledge with me during our scientific discussions and later with the manuscript. I would
also like to mention the fantastic welcome that I had with your fantastic research group. I was very
pleased with my days in Ljubljana, and I will never forget this experience.
… to Dr. Bojana Žegura, for the direct laboratory supervision, precise guidelines, scientific
accuracy and friendship.
… to Senhora Engenheira Elisa and all the staff from the Laboratory of Bromatology and
Hidrology of Faculty of Pharmacy – University of Porto for the assistance they gave me during this
period, especially to Professor Doctor Susana Casal for all scientific support.
… to all collaborators in my PhD work, especially to Paula, Edgar, Marko, Armindo and Luis
Filipe.
… to my friends Joana Santos and Cristina Soares, for your friendship, hospitality, and for help me
in all kinds of situations during my PhD.
… to my friends Diana, Vitor, Zé, João Bernardo, Alja, Javor, Kjell, Heryka, Ivone, Telmo,
Anabela C. and Sónia, for all the moments!
… to my friends from Figueira, you make my life so much funnier!
… to EA, since I met you, everything was much happier and easier. Thank you for being there.
… to my dad, mother, Marta and Cristiana, for your support in all my life. I am proud of my
principles, strength and character and all these came from you. There are no words to thank you.
And I can not forget to express my gratitude to Tia Rosa for watching over us.
… to God, for your protection and because in the end everything always worked out.

vii
Abstract

Heterocyclic aromatic amines (HAs) are considered a dietary risk factor for human cancer. Their
capability of formation on muscle foods during ordinary cooking practices (grilling, broiling,
barbecuing, roasting, frying, pan-frying), even at low parts-per-billion (ppb), implies frequent
exposure by the general public. Over the past 30 years, numerous studies have been stimulated
aiming to alleviate human health risk associated with HAs. The three main areas are still a
challenge: their occurrence in foods, the strategies to inhibit their formation, and the search for
chemopreventive agents. Furthermore, several researchers highlighted an urgent need of studying
HAs and other concomitant mutagens at the same time, as polycyclic aromatic hydrocarbons
(PAHs).
The occurrence of HAs and PAHs in different cooked muscle foods (beef, salmon, and sardines)
and different cooking procedure (barbecuing, grilling and pan-frying) on Portuguese household
cooking procedures were evaluated. The samples were analyzed for HAs contents using a reference
method, which consists in solid-phase extraction (SPE) and high-performance liquid
chromatography-diode array detection/ fluorescence detection (HPLC-DAD/FLD). To PAHs a
similar methodology was ascertained, which also consists in SPE and separation and detection by
HPLC-FLD.
Concerning the HAs formation in analyzed samples, in general the most abundant HA was PhIP
(1.45-33.8 ng/g), followed by AαC (1-19 ng/g) and MeIQx (not detected-9.07ng/g). The HAs, IQ,
4,8-DiMeIQx, MeAαC, Trp-P-1, Trp-P-2 and Glu-P-1 were also formed in some muscle foods and
cooking procedures with relative significance. In contrast IQx, MeIQ, 7.8-DiMeIQx, 4,7,8-
TriMeIQx, and Glu-P-2 were not detected at all. Thermic HAs were frequently studied, whereas
pyrolytic HAs were usually neglected. However, the contribution of AαC is crucial in HAs
exposition. In relation to PAHs occurrence in grilled muscle foods, quantitative PAHs profiles were
different among salmon> chicken> beef. PAH8 content is significantly correlated with fat content
of these muscle foods.
The cooking conditions affect the formation of HAs and PAHs in muscle foods. Efficient strategies
on the reduction of HAs in fatty fish were grilling at longer distance from charcoal or substitute the
wood charcoal by coconut shell charcoal or use an electrical grill. The continuous barbecuing with
the same charcoal contributed to higher formation of HAs and PAHs.
The most promising mitigation strategies on HAs are the addition of antioxidant compounds. The
inhibitory effect of antioxidant rich marinades was evaluated in pan-fried beefs. Beer as marinade
applied 4 hours prior to cook the beefs, inhibited around 80% of the total HAs formation.
Considering the inhibitory effect of beer as the standard, the relative efficiencies of other liquid
marinades on the total HAs formation were: 1 to beer, 0.9 to dealcoholized white wine, 0.7 to red
viii
wine, 0.7 to white wine and 0.6 to green tea marinade. A mixture of spices (herbs) commonly used
as meat flavoring (garlic, ginger, thyme, rosemary and red chili pepper) was also evaluated in
combination with the liquid marinades (beverages). Relative efficiencies were: 1.1 to beer with
herbs, 0.95 to dealcoholized white wine with herbs, 0.6 to white wine with herbs. Herbs explained
around 30% of inhibition of PhIP formation, in contrast alcohol seems to perform a strong
influence on PhIP formation. No correlation was observed between antiradical activity of
marinades and total or individual HAs formation.
Xanthohumol (XN) is a hop compound contained in beer, described as a “broad-spectrum”
chemopreventive. In the present study XN completely prevented the HAs (PhIP and MeIQx)
induced DNA strand breaks at nanomolar concentrations, in HepG2 cells. QRT-PCR gene
expression analysis of the main enzymes involved in the biotransformation revealed that the
protective effect of XN against HAs may be mediated by up-regulation of UGT1A1 expression.
Beer or their compounds can prevent HAs carcinogenesis, either by inhibiting their formation or
also as chemopreventing agent.

ix
Resumo

As aminas aromáticas heterocíclicas (HAs) são consideradas um fator de risco para o
desenvolvimento de cancro. A capacidade de formação das HAs em carnes e pescado cozinhados
pelos métodos culinários habituais (fritos, assados, grelhados na frigideira, no carvão ou em
dispositivo elétrico), mesmo em quantidades muito pequenas (ppb), implicam uma frequente
exposição pela população em geral. Nos últimos 30 anos, realizaram-se inúmeros estudos visando a
minimização do risco para a saúde associado às HAs. Contudo, as três principais áreas de estudo
das HAs ainda são um desafio: a formação em alimentos, as estratégias de inibição da formação, e
a procura de compostos quimiopreventivos. Além disso, vários investigadores têm realçado a
necessidade do estudo de outros carcinogénios que ocorram em simultâneo com as HAs, como os
hidrocarbonetos policíclicos aromáticos (PAHs).
A presença de HAs e PAHs foi avaliada em carnes (bife e frango) e pescado (salmão e sardinha)
cozinhados de diferentes formas, refletindo as práticas portuguesas. O conteúdo em HAs nas
amostras foi determinado usando um método de referência, que consiste em extração e purificação
por extração em fase sólida (SPE) e separação e deteção por cromatografia líquida de alta
eficiência acoplada a sistemas de deteção por díodos e fluorescência (HPLC-DAD/FLD). Os PAHs
foram analisados através de uma metodologia determinada na presente dissertação, que consiste
também em extração e purificação por SPE e separação e deteção por HPLC-FLD.
Relativamente à formação de HAs nas amostras analisadas, em geral, a HA mais abundante foi o
PhIP (1,45-33,8 ng/g), seguido de AαC (1-19 ng/g) e MeIQx (não detectado-9,07ng/g). A formação
das HAs IQ, 4,8-DiMeIQx, MeAαC, Trp-P-1, Trp-P-2 e Glu-P-1 verificou-se em algumas amostras
em função do tipo de músculo ou método culinário. Em contrapartida as HAs IQx, MeIQ, 7.8-
DiMeIQx, 4,7,8-TriMeIQx, e Glu-P-2 não foram detetadas em nenhuma das amostras em estudo.
Na literatura verifica-se que as HAs térmicas são frequentemente estudadas, enquanto as HAs
pirolíticas são normalmente negligenciadas. Contudo, a contribuição do AαC, HA pirolítica, é
crucial para a avaliação da exposição às HAs. Em relação aos PAHs, observaram-se diferenças no
perfil quantitativo entre as amostras: salmão> frango> bife. Verificou-se que o conteúdo em PAH8
está correlacionado significativamente com o teor de gordura da amostra.
Alterar as condições de confeção pode afetar a formação de HAs e PAHs em carnes e pescado.
Relativamente aos peixes gordos grelhados, a redução da formação de HAs verificou-se ao grelhar
o peixe a maior distância do carvão, ao substituir o carvão vegetal tradicional por carvão de casca
de coco e através do uso do grelhador elétrico. Constatou-se ainda que grelhar em carvão de casca
de coco também reduz a formação de PAHs em salmão. O uso contínuo do mesmo carvão na
preparação de frango de churrasco contribui para uma maior quantidade de HAs e PAHs neste
alimento.
x