Master of Science / Department of Food Science / J. Scott Smith / Heterocyclic amines (HCAs) formed during cooking of meats at high temperatures are suspected cancer causing compounds and efforts have been made to reduce their levels. Spices possessing high levels of antioxidants have been shown to inhibit these compounds when incorporated prior to cooking. Seven spices, black pepper, rosemary, turmeric, thyme, cinnamon, ginger and oregano were analyzed for their antioxidant capacity using three different assays. These spices were individually added at 0.25% to beef patties fried at 400 °F for 5 min per side to evaluate their effect on HCA inhibition. Black pepper was emphasized in this study by studying the effect of addition at different concentrations (0.25, 0.50, 1.00 %) on HCA reduction. It was found that patties treated with black pepper individually, as well in combination with other spices, greatly decreased the formation of HCAs. Black pepper at 0.25% level showed the highest inhibition of PhIP levels (85%). The spices were shown to have high levels of free radical scavenging activity as measured by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. Overall, black pepper had significantly lower DPPH scavenging activity but still showed high HCA inhibition. All the spices individually inhibited HCA by 55-82%. Black pepper in combination with turmeric was the best combination, showing a 94.74% inhibition of HCA formation.

Master of Science / Food Science Institute / J. Scott Smith / Heterocyclic amines (HCAs) are a class of toxicological compounds that can be formed during heating of precursors, amino acids, creatinine, creatine, and sugars at high temperature cooking of muscle products. These potent mutagens are suspected to play a role in human cancers. The objective of this study was to investigate a practical method to reduce the amount of HCAs through marinating of beef steaks. We were interested in the potential health benefits of natural extracts containing polyphenols present in commercial marinades. HCAs were compared in marinated and unmarinated steaks. Four common HCAs were investigated: 2-amino-3,8- dimethyl-imidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenyl-imidazo[4,5- b]pyridine (PhIP), 1-methyl-9H-pyrido[4,3-b]-indol (harman), and 9H-pyrido[4,3-b]-indol (norharman). Steaks were marinated for one hour and grilled at 400 °F for 5 minutes at each side. Meat samples were extracted by solid phase extraction (SPE) and analysis with HPLC showed the significant decrease (p < 0.05) of sum of polar and nonpolar HCAs by 71% compared to untreated steaks. For confirmation of spices potency on reduction of HCA formation, the same experiment was applied to meat with the base of commercial marinade powders excluding the herbs and spices. Lesser reduction of HCAs were shown and in some cases no significant reduction occurred. HPLC analysis showed presence of considerable amount of natural phenolic antioxidants of carnosic acid, carnosol, and rosmarinic acid, which can be related to the reduction effects of HCA formation in commercial marinades. These results revealed that marinating meats before grilling with various spices/herbs containing antioxidants may reduce formation of mutagenic /carcinogenic HCAs markedly.

Master of Science / Food Science Institute / J. Scott Smith / Heterocyclic amines (HCAs) are potentially carcinogenic and highly mutagenic byproducts of the Maillard browning reaction that form specifically in high temperature cooked meat products. Consumption of HCAs has been associated with various cancers including prostate, breast, colon, and pancreatic cancers and efforts have been made to understand formation and inhibition of these compounds. Chemical model systems are a preferred method to study the in vitro formation and inhibition of HCAs as the complex matrix effects found in meat are eliminated. Two black pepper extracts were evaluated for their efficacy on PhIP formation in model systems, but no significant results were observed. Secondly, four Maillard reaction variables were evaluated for their effect on formation of five HCAs (IQ, IQx, MeIQ, MeIQx, and 4,8-DiMeIQx) in chemical model systems with an effort to define an ideal model system. Precursor molar concentration (0.2/0.2, 0.4/0.4, 0.6/0.6, and 0.8/0.8 mmol), water percentage (0, 5, 10, and 15%), sugar type (fructose, galactose, glucose, and lactose), and sugar molar amount (quarter, half, equi, and double molar) were the four Maillard variables examined in the study. Additionally, four antioxidants (butylated hydroxyanisole (BHA), epigallocatechin gallate (EGCG), rosmarinic acid, and naringenin) were evaluated for their effect on HCA formation in chemical model systems. All four Maillard variables had a significant effect (p < 0.05) on the formation of HCAs in the model system, with an interaction effect occurring between water percentage and precursor concentration. The four antioxidants had no effect on the formation of HCAs in the model system. A model system containing 0.6/0.6/1.2 mmol of threonine, creatinine, and glucose, with 15% water was determined to be the best representative chemical model system for the formation of HCAs commonly formed in meats.

Doctor of Philosophy / Food Science Institute -- Animal Science & Industry / J. Scott Smith / Heterocyclic amines (HCAs) are produced in meats cooked at high temperature, which are potent mutagens and a risk factor for human cancers. Occurrence of HCAs in ready-to-eat (RTE) meat products and cooked meat products based on prevalence of various cooking methods that are preferred among U.S. meat consumers were investigated. The primary HCAs detected in samples were PhIP (2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine), MeIQx (2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline), and DiMeIQx (2-amino-3,4,8-trimethyl-imidazo [4,5-f]quinoxaline). RTE meat products were ranked in the following order of increasing total HCA content: pepperoni (0.05 ng/g) < hot dogs and deli meat products (0.5 ng/g) < fully cooked bacon (1.1 ng/) < rotisserie chicken meat (1.9 ng/g) < rotisserie chicken skin (16.3 ng/g). In cooked meat products, high levels of total HCAs were found in fried pork (13.91 ng/g), fried fish (14.91 ng/g), and fried bacon (17.91 ng/g). Inhibition of HCAs by rosemary extracts, which were extracted with different solvents, were evaluated in cooked beef patties. Five rosemary extracts were 100W (100% water), 10E (10% ethanol), 20E (20% ethanol), 30E (30% ethanol), and 40E (40% ethanol). Rosemary extract 20E containing a mixture of rosmarinic acid (27.3 mg/g), carnosol (72.9 mg/g), and carnosic acid (4.2 mg/g) showed the greatest inhibition of MeIQx (up to 91.7%) and PhIP (up to 85.3%). The effect of enhancement and marination on HCA formation in meat products was investigated. The addition of salt and phosphate greatly improved the water-holding capacity and decreased HCA formation (up to 58%) in enhanced fresh meat products. An greater reduction of HCAs (up to 79%) was found in marinated fresh meat; the enhancement solution for this meat contained ingredients that exhibited good antioxidant properties.

When food is heated, a vast number of compounds are formed. Some of these are known to be toxic. Among these are furan, HMF, PhIP, IQ, and MeIQx, the subjects of this thesis. All these compounds are known or suspected carcinogens but the detailed mechanisms behind their carcinogenicity have not yet been fully examined. The aim of this thesis was to study genotoxic properties of these compounds using both in vitro and in vivo methods. Clastogenic effects of all five compounds were assessed with the flow cytometer-based micronucleus assay in vivo and for furan also with the micronucleus assay in vitro. DNA-damaging effects of HMF were studied using the comet assay. No induction of micronuclei was obtained after exposure to IQ, MeIQx or furan. Hence, it can be argued that non-genotoxic mechanisms are partly responsible for the carcinogenic properties of these compounds. PhIP, on the other hand, generated a clear response in the in vivo test. Comparing these result with previous results on acrylamide indicates that PhIP is much more potent. However, acrylamide probably poses a higher risk for humans as the intake is considerably higher. For HMF no effects were seen using the in vivo setup. To further investigate the influence of bioactivation of HMF by sulfotransferases (SULTs) the comet assay was performed in cell lines expressing different levels of SULT. However, no correlation between SULT-expression and DNA-damage was observed. Thus, the DNA-damaging effects found in our experimental setup is probably due to other factors than SULT mediated effects. Furthermore, in this thesis the effects of folic acid on chromosomal stability in healthy people were studied. A negative correlation was found between micronucleus frequency and folate status. The results gained within this thesis will hopefully contribute to the risk assessment of compounds present in our diet.

We have investigated phase II activation of the food-derived mutagen 2- hydroxyamino-1-methyl-6-phenyl[4,5-b]pyridine (N-OH-PhIP) by cytosolic acetyltransferase, sulfotransferase, and tRNA synthetase/kinase enzymes from human breast tissue. Cytosol from homogenates of mammary gland tissue obtained from breast-reduction surgery or mastectomy was incubated with and without enzyme-specific cofactors, and mutagen binding to calf thymus DNA was quantified by 32P-postlabeling. In addition, microsomal fractions of mammary epithelial cells from some individuals were examined for prostaglandin H synthetase activation of N-OH-PhIP. Our results show that all four enzymes can participate in activating N-OH-PhIP, thus inducing PhIP-DNA adduct formation in human mammary cells. However, not all individuals exhibited all these activities; instead each individual showed a combination of one or more activation pathways. The present findings demonstrate that the human mammary gland has the capacity to metabolically activate a dietary mutagen by several enzyme systems, including acetyltransferase, sulfotransferase, tRNA synthetase/kinase, and prostaglandin hydroperoxidase catalysis.

Mutagenic heterocyclic amines are produced during the ordinary cooking of meats and fish. When metabolically activated, heterocyclic amines will form covalent adducts with DNA, which, if not repaired, may affect the flow of genetic information in a cell. It has been proposed previously that heterocyclic amine mutagens contribute to the incidence of dietary-related cancers because they cause somatic cell mutations and induce tumors in rodents and nonhuman primates. Recent work has shown that some cooked food mutagens preferentially produce DNA damage in heart cells, DNA adduct levels are directly related to dose and duration of mutagen exposure, the dietary damage persists for long intervals in cardiac tissue, and mitochondrial DNA is more vulnerable than nuclear DNA to these mutagens. Because cardiac myocytes are terminally differentiated cells that have lost their ability to divide, the capacity to repair DNA damage is a critical factor in the proper function of cardiomyocytes, and cardiac myocytes seem to have limited DNA repair capabilities. DNA damage formed by dietary components, such as heterocyclic amines, might accumulate with time because of inefficient repair and thereby affect heart cell function or viability. The possibility that dietary habits play a role in idiopathic cardiomyopathies and congestive heart disease should be explored in greater depth.

Introdução. O alto consumo de carne, principalmente vermelha e processada, tem sido relacionado com aumento de risco de doenças crônicas, especialmente o câncer. Uma das explicações possíveis são os métodos de preparo culinário a altas temperaturas, que acarretam na formação aminas heterocíclicas. Estes compostos são detoxificados no nosso organismo, passando por um processo, no qual podem ser geradas espécies reativas, relacionadas ao estresse oxidativo e ao dano ao DNA. Entretanto, os indivíduos apresentam respostas diferentes à mesma exposição dietética, podendo ter diferentes níveis de risco ou benefício com a mesma ingestão de alimentos. O código genético individual pode ser uma das causas dessa variação interpessoal. Objetivo. Investigar a relação entre o consumo de carnes e aminas heterocíclicas com estresse oxidativo e dano no DNA, considerando polimorfismos genéticos, fatores demográficos e de estilo de vida em residentes do Município de São Paulo. Métodos. Foram utilizados dados dietéticos, genéticos, bioquímicos e estilo de vida de um estudo transversal com amostra probabilística de múltiplo estágio chamado Inquérito de Saúde de São Paulo (ISACapital). Os dados de carne e aminas heterocíclicas foram obtidos a partir de um recordatório alimentar de 24 horas e questionário sobre métodos de cocção e graus de cozimento das carnes. A extração do DNA ocorreu pelo método por sal e utilizou-se a técnica PCR em tempo real para determinação dos seguintes polimorfismos de nucleotídeo único: CYP1A1 (rs1048943), CYP1A2 (rs762551, rs35694136), CYP1B1 (rs1056836, rs10012), NAT2 (rs1208, rs1041983, rs1799929, rs1801280, rs1799931, rs1799930, rs1801279), NAT1 (rs4986782, rs5030839, rs56379106, rs56318881, rs6586714), SULT1A1 (rs928286), UGT1A9 (rs3832043), SOD2 (rs4880), CAT (rs7943316), GSTA1 (rs3957357), GSTP1 (rs1695), e deleção dos genes GSTM1 e GSTT1. Foram utilizados os biomarcadores malonaldeído (MDA) no plasma para estimar o estresse oxidativo e o 8-OHdG no plasma para estimar dano ao DNA. As associações foram examinadas por meio de modelos de regressão múltipla linear e logística ajustadas por sexo, idade, IMC, consumo de frutas e calorias, atividade física e fumo. Resultados. O consumo médio de aminas heterocíclicas foi de 437ng/dia e a carne de boi foi a que mais contribuiu para o consumo de aminas. Participantes que consumiram carne de boi grelhada muito bem passada apresentaram maiores concentrações de MDA do que os demais. Encontrou-se associação positiva entre consumo de aminas heterocíclicas com estresse oxidativo e dano ao DNA, isto é, indivíduos que consumiram maiores teores de aminas heterocíclicas apresentaram maiores chances de ter elevados concentrações de MDA (OR=1,17; P=0,04) e maiores concentrações de 8-OHdG (=1,62; P=0,04). Observou-se também que esta associação pode ser modificada pelas características genéticas individuais, sendo que polimorfismos nos genes das enzimas de detoxificação NAT2 e CYP1B1 interagiram com o consumo de aminas, diminuindo o estresse oxidativo. Conclusão. Verificou-se que o alto consumo de aminas heterocíclicas contribuiu para maiores níveis de estresse oxidativo e dano ao DNA independente de fatores demográficos e de estilo de vida, aumentando o risco de doenças crônicas. Observou-se também que esta relação pode ser alterada na presença de polimorfismos genéticos individuais. / Introduction. The excessive meat intake, especially red and processed meat, has been linked to chronic diseases, especially cancer. One of the reasons for that is the cooking process at high temperatures that can form heterocyclic amines (HCA). During HCA metabolism, reactive species can be formed, which can cause oxidative stress and DNA damage. However, people can show different answers to the same food intake, increasing or decreasing the risk of diseases. The DNA code can be one of the causes of this between-person variations. Objective. To investigate the association between meat/heterocyclic amine intake with oxidative stress and DNA damage, considering polymorphism, demographic and life style factors among population of São Paulo city. Methods. Information on food intake, genetics, biochemical, and lifestyle was obtained from a representative, multistage probability-based cross-sectional study titled Health Survey for Sao Paulo (ISA-Capital). Meat and heterocyclic amine intake was estimated by a 24-hour dietary recall complemented by a detailed questionnaire with preferences of cooking methods and level of doneness for meats. The salt method was used for DNA extraction and real time PCR to identify the following single nucleotide polymorphisms: CYP1A1 (rs1048943), CYP1A2 (rs762551, rs35694136), CYP1B1 (rs1056836, rs10012), NAT2 (rs1208, rs1041983, rs1799929, rs1801280, rs1799931, rs1799930, rs1801279), NAT1 (rs4986782, rs5030839, rs56379106, rs56318881, rs6586714), SULT1A1 (rs928286), UGT1A9 (rs3832043), SOD2 (rs4880), CAT (rs7943316), GSTA1 (rs3957357), GSTP1 (rs1695), GSTM1 and GSTT1 (null or not). We used malondialdehyde (MDA) concentration in plasma to estimated oxidative stress, and 8-OHdG concentration in plasma to estimate DNA damage. Analyses were performed using multivariate logistic and linear regressions adjusted for smoking, sex, age, body mass index, energy intake, fruit intake, smoking and physical activity. Results. Mean HCA intake was 437ng/day and beef was the meat that contributed more to HCA. Participants who consumed grilled beef very well-done presented more MDA concentration than other participants. We found significant association between heterocyclic amine intake with oxidative stress and DNA damage. Participants who consumed high levels of heterocyclic amines showed higher odds to show high MDA concentration (OR=1.17; P=0.04) and high 8-OHdG concentration (=1.62; P=0.04). These associations could be modified by individual genetic characteristics. Polymorphisms in genes that codify NAT2 and CYP1B1 detoxification enzymes interacted with HCA intake, decreasing oxidative stress. Conclusions. The high heterocyclic amine intake contributed to increase oxidative stress independently of lifestyle and demographic factors, increasing risk of chronic diseases. These relationships can be modified by genetic polymorphisms.

As aminas heterocíclicas alifáticas, piperidina, piperazina e pirrolidina, interagem com o dióxido de enxofre, gasoso ou líquido, dando origem a uma série de compostos em diferentes estequiometrias, os quais foram investigados fundamentalmente por técnicas espectroscópicas vibracionais, infravermelho e Raman, utilizando-se como técnicas auxiliares a espectroscopia eletrônica, espectros de massas e de ressonância magnética nuclear. Os resultados obtidos caracterizam as espécies estudadas como complexos moleculares formados entre as aminas e o SO2, especialmente devido aos deslocamentos de frequências observados nos espectros Raman, para os modos vibracionais de estiramento simétrico e deformação angular do dióxido de enxofre. Os dados indicam no sentido do estabelecimento de intensas ligações de hidrogênio, as quais devem desempenhar importante papel na estabilização dos complexos. Os deslocamentos observados nos espectros Raman encontram-se de acordo com o comportamento previsto para a interação de transferência de carga, em que o LUMO do SO2 apresenta caráter antiligante em relação à ligação S-O e ligante em relação à interação O-O. / The interaction of alifatic heterocyclic amines piperidine, piperazine and pyrrolidine, with sulfur dioxide yields a variety of products of different stoichiometry. The investigation of the formed species were carried out mainly by vibrational spectroscopic techniques, infrared and Raman, and electronic spectroscopy, mass spectrometry and nuclear magnetic resonance spectra were used as auxiliary techniques. The obtained results indicate that the studied species are charge transfer molecular complexes due to the frequency shifts of the symmetric stretching and angular deformation modes of sulfur dioxide in the Raman spectra. The data are in agreement with an S-O antibonding and a O-O bonding character of the LUMO of SO2. Hydrogen bonds play an important role in the complex stabilization.

As aminas heterocíclicas alifáticas, piperidina, piperazina e pirrolidina, interagem com o dióxido de enxofre, gasoso ou líquido, dando origem a uma série de compostos em diferentes estequiometrias, os quais foram investigados fundamentalmente por técnicas espectroscópicas vibracionais, infravermelho e Raman, utilizando-se como técnicas auxiliares a espectroscopia eletrônica, espectros de massas e de ressonância magnética nuclear. Os resultados obtidos caracterizam as espécies estudadas como complexos moleculares formados entre as aminas e o SO2, especialmente devido aos deslocamentos de frequências observados nos espectros Raman, para os modos vibracionais de estiramento simétrico e deformação angular do dióxido de enxofre. Os dados indicam no sentido do estabelecimento de intensas ligações de hidrogênio, as quais devem desempenhar importante papel na estabilização dos complexos. Os deslocamentos observados nos espectros Raman encontram-se de acordo com o comportamento previsto para a interação de transferência de carga, em que o LUMO do SO2 apresenta caráter antiligante em relação à ligação S-O e ligante em relação à interação O-O. / The interaction of alifatic heterocyclic amines piperidine, piperazine and pyrrolidine, with sulfur dioxide yields a variety of products of different stoichiometry. The investigation of the formed species were carried out mainly by vibrational spectroscopic techniques, infrared and Raman, and electronic spectroscopy, mass spectrometry and nuclear magnetic resonance spectra were used as auxiliary techniques. The obtained results indicate that the studied species are charge transfer molecular complexes due to the frequency shifts of the symmetric stretching and angular deformation modes of sulfur dioxide in the Raman spectra. The data are in agreement with an S-O antibonding and a O-O bonding character of the LUMO of SO2. Hydrogen bonds play an important role in the complex stabilization.