Kinetic models as a route to control acrylamide formation in food

Reports that heat processing of foods induces the formation of acrylamide heightened interest in the chemistry, biochemistry, and safety of this compound. Acrylamide-induced neurotoxicity, reproductive toxicity, genotoxicity, and carcinogenicity are potential human health risks based on animal studies. Because exposure of humans to acrylamide can come from both external sources and the diet, there exists a need to develop a better understanding of its formation and distribution in food and its role in human health. To contribute to this effort, experts from eight countries have presented data on the chemistry, analysis, metabolism, pharmacology, and toxicology of acrylamide. Specifically covered are the following aspects: exposure from the environment and the diet; biomarkers of exposure; risk assessment; epidemiology; mechanism of formation in food; biological alkylation of amino acids, peptides, proteins, and DNA by acrylamide and its epoxide metabolite glycidamide; neurotoxicity, reproductive toxicity, and carcinogenicity; protection against adverse effects; and possible approaches to reducing levels in food. Cross-fertilization of ideas among several disciplines in which an interest in acrylamide has developed, including food science, pharmacology, toxicology, and medicine, will provide a better understanding of the chemistry and biology of acrylamide in food, and can lead to the development of food processes to decrease the acrylamide content of the diet.

Genistein protects human mammary epithelial cells from benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide and 4-hydroxy-2-nonenal genotoxicity by modulating the glutathione/glutathione S-transferase system

Epidemiological studies have shown that ingestion of isoflavone-rich soy products is associated with a reduced risk for the development of breast cancer. In the present study, we investigated the hypothesis that genistein modulates the expression of glutathione S-transferases (GSTs) in human breast cells, thus conferring protection towards genotoxic carcinogens which are GST substrates. Our approach was to use human mammary cell lines MCF-10A and MCF-7 as models for non-neoplastic and neoplastic epithelial breast cells, respectively. MCF-10A cells expressed hGSTA1/2, hGSTA4-4, hGSTM1-1 and hGSTP1-1 proteins, but not hGSTM2-2. In contrast, MCF-7 cells only marginally expressed hGSTA1/2, hGSTA4-4 and hGSTM1-1. Concordant to the protein expression, the hGSTA4 and hGSTP1 mRNA expression was higher in the non-neoplastic cell line. Exposure to genistein significantly increased hGSTP1 mRNA (2.3-fold), hGSTP1-1 protein levels (3.1-fold), GST catalytic activity (4.7-fold) and intracellular glutathione concentrations (1.4-fold) in MCF-10A cells, whereas no effects were observed on GST expression or glutathione concentrations in MCF-7 cells. Preincubation of MCF-10A cells with genistein decreased the extent of DNA damage by 4-hydroxy-2-nonenal (150 mu M) and benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (50 mu M), compounds readily detoxified by hGSTA4-4 and hGSTP1-1. In conclusion, genistein pretreatment protects non-neoplastic mammary cells from certain carcinogens that are detoxified by GSTs, suggesting that dietary-mediated induction of GSTs may be a mechanism contributing to prevention against genotoxic injury in the aetiology of breast cancer.

Investigations on the effect of amino acids on acrylamide, pyrazines, and Michael addition products in model systems

Acrylamide and pyrazine formation, as influenced by the incorporation of different amino acids, was investigated in sealed low-moisture asparagine-glucose model systems. Added amino acids, with the exception of glycine and cysteine and at an equimolar concentration to asparagine, increased the rate of acrylamide formation. The strong correlation between the unsubstituted pyrazine and acrylamide suggests the promotion of the formation of Maillard reaction intermediates, and in particular glyoxal, as the determining mode of-action. At increased amino acid concentrations, diverse effects were observed. The initial rates of acrylamide formation remained high for valine, alanine, phenylalanine, tryptophan, glutamine, and Ieucine, while a significant mitigating effect, as evident from the acrylamide yields after 60 min of heating at 160 degrees C, was observed for proline, tryptophan, glycine, and cysteine. The secondary amine containing amino acids, proline and tryptophan, had the most profound mitigating effect on acrylamide after 60 min of heating. The relative importance of the competing effect of added amino acids for alpha-dicarbonyls and acrylamide-amino, acid alkylation reactions is discussed and accompanied by data on the relative formation rates of selected amino acid-AA adducts.

Acrylamide and pyrazine formation in model systems containing asparagine

The effect of different sugars and glyoxal on the formation of acrylamide in low-moisture starch-based model systems was studied, and kinetic data were obtained. Glucose was more effective than fructose, tagatose, or maltose in acrylamide formation, whereas the importance of glyoxal as a key sugar fragmentation intermediate was confirmed. Glyoxal formation was greater in model systems containing asparagine and glucose rather than fructose. A solid phase microextraction GC-MS method was employed to determine quantitatively the formation of pyrazines in model reaction systems. Substituted pyrazine formation was more evident in model systems containing fructose; however, the unsubstituted homologue, which was the only pyrazine identified in the headspace of glyoxal-asparagine systems, was formed at higher yields when aldoses were used as the reducing sugar. Highly significant correlations were obtained for the relationship between pyrazine and acrylamide formation. The importance of the tautomerization of the asparagine-carbonyl decarboxylated Schiff base in the relative yields of pyrazines and acrylamide is discussed.

Fecal water as a non-invasive biomarker in nutritional intervention: comparison of preparation methods and refinement of different endpoints

The assessment of cellular effects by the aqueous phase of human feces (fecal water, FW) is a useful biomarker approach to study cancer risks and protective activities of food. In order to refine and develop the biomarker, different protocols of preparing FW were compared. Fecal waters were prepared by 3 methods: (A) direct centrifugation; (B) extraction of feces in PBS before centrifugation; and (C) centrifugation of lyophilized and reconstituted feces. Genotoxicity was determined in colon cells using the Comet assay. Selected samples were investigated for additional parameters related to carcinogenesis. Two of 7 FWs obtained by methods A and B were similarly genotoxic. Method B, however, yielded higher volumes of FW, allowing sterile filtration for long-term culture experiments. Four of 7 samples were non-genotoxic when prepared according to all 3 methods. FW from lyophilized feces and from fresh samples were equally genotoxic. FWs modulated cytotoxicity, paracellular permeability, and invasion, independent of their genotoxicity. All 3 methods of FW preparation can be used to assess genotoxicity. The higher volumes of FWobtained by preparation method B greatly enhance the perspectives of measuring different types of biological parameters and using these to disclose activities related to cancer development.

Gut fermentation products of insulin-derived prebiotics beneficially modulate markers of tumour progression in human colon tumour cells

Insulin is a prebiotic food ingredient, which suppresses colon tumour growth and development in rats. In the gut lumen, it is fermented to lactic acid and short chain fatty acids (SCFA). Of these, butyrate has suppressing agent activities, but little is known concerning cellular responses to complex fermentation samples. To investigate the effects of fermentation products of insulin on cellular responses related to colon carcinogenesis. Fermentations were performed in anaerobic batch cultures or in a three-stage fermentation model that simulates conditions in colon-segments (proximal, transverse, distal). Substrate was insulin enriched with oligofructose (Raftilose® Synergy1), fermented with probiotics (Bifidobacterium lactis Bb12, Lactobacillus rhamnosus GG), and/or faecal inocula. HT29 or CaCo-2 cells were incubated with supernatants of the fermented samples (2.5%-25% v/v, 24-72 hours). Cellular parameters of survival, differentiation, tumour progression, and invasive growth were determined. Fermentation supernatants derived from probiotics and Synergy1 were more effective than with glucose. The additional fermentation with faecal slurries produced supernatants with lower toxicity, higher SCFA contents, and distinct cellular functions. The supernatant derived from the gut model vessel representing the distal colon, was most effective for all parameters, probably on account of higher butyrate-concentrations. Biological effects of insulin upon colon cells may be mediated not only by growth stimulation of the lactic acid-producing bacteria and/or production of butyrate, but also by other bacteria and products of the gut lumen. These newly reported properties of the supernatants to inhibit growth and metastases in colon tumour cells are important mechanisms of tumour suppression.

Modulation of detoxification enzymes by watercress: in vitro and in vivo investigations in human peripheral blood cells

Epidemiological studies indicate that consumption of cruciferous vegetables (CV) can reduce the risk of cancer. Supposed mechanisms are partly the inhibition of phase I and the induction of phase II enzymes. The aim of this study was to investigate in vitro and in vivo effects of watercress (WC), a member of the CV family, on chemopreventive parameters using human peripheral blood mononuclear cells (PBMC) as surrogate cells. We investigated the hypothesis that WC reduces cancer risk by inducing detoxification enzymes in a genotype-dependent manner. In vitro gene expression and enzyme activity experiments used PBMC incubated with a crude extract from fresh watercress (WCE, 0.1-10 mu L/mL with 8.2 g WC per 1 mL extract) or with one main key compound phenethyl isothiocyanate (PEITC, 1-10 mu M). From an in vivo perspective, gene expression and glutathione S-transferase (GST) polymorphisms were determined in PBMC obtained from a human intervention study in which subjects consumed 85 g WC per day for 8 weeks. The influence of WC consumption on gene expression was determined for detoxification enzymes such as superoxide dismutase 2 (SOD2) and glutathione peroxidase 1 (GPX1), whilst the SOD and GPX activities in red blood cells were also analysed with respect to GST genotypes. In vitro exposure of PBMC to WCE or PEITC (24 h) increased gene expression for both detoxification enzymes GPX1 (5.5-fold, 1 mu L/mL WCE, 3.7-fold 1 mu M PEITC) and SOD2 (12.1-fold, 10 mu L/mL WCE, 7.3-fold, 10 mu M PEITC), and increased SOD2 activity (1.9-fold, 10 mu L/mL WCE). The WC intervention had no significant effect on in vivo PBMC gene expression, as high individual variations were observed. However, a small but significant increase in GPX (p = 0.025) and SOD enzyme activity (p = 0.054) in red blood cells was observed in GSTM1*0, but not in GSTM1*1 individuals, whilst the GSTT1 genotype had no impact. The results indicate that WC is able to modulate the enzymes SOD and GPX in blood cells in vitro and in vivo, and suggest that the capacity of moderate intake of CV to induce detoxification is dependent in part on the GSTM1 genotype.

The effect of cooking on acrylamide and its precursors in potato, wheat and rye

The relationship between acrylamide and its precursors, namely free asparagine and reducing sugars, was studied in simple cakes made from potato flake, wholemeal wheat and wholemeal rye, cooked at 180 degrees C, from 5 to 60 min. Between 5 and 20 min, large losses of asparagine, water and total reducing sugars were accompanied by large increases in acrylamide, which maximized in all three products between 25 and 30 min, followed by a slow linear reduction. Acrylamide formation did not occur to any extent until the moisture contents of the cakes fell below 5%. A comparison of each type of cake with a commercial product, made from the same food material, showed that acrylamide levels in all three commercial products were well below the maximum levels in the cooked cakes.

Measurement of acrylamide and its precursors in potato, wheat, and rye model systems

The relationship between acrylamide and its precursors, namely, free asparagine and reducing sugars, was studied in cakes made from potato flake, wholemeal wheat, and wholemeal rye, cooked at 180 degreesC, from 5 to 60 min. Between 5 and 20 min, major losses of asparagine, water, and total reducing sugars were accompanied by large increases in acrylamide, which maximized in all three products between 25 and 30 min, followed by a slow linear reduction. Acrylamide formation did not occur to a large degree until the moisture contents of the cakes fell below 5%. Linear relationships were observed for acrylamide formation with the residual levels of asparagine and reducing sugars for all three food materials.

The effects of alpha-lactalbumin and glycomacropeptide on the association of CaCo-2 cells by enteropathogenic Escherichia coli, Salmonella typhimurium and Shigella flexneri

Two milk components, alpha-lactalbumin (alpha-La) and glycomacropeptide (GMP) may inhibit intestinal infection/intoxification. (3)[H] thymidine-labeled enteropathogenic Escherichia coli (EPEC), Salmonella typhimurium (ATCC 6994) or Shigella flexneri (ATCC 9199) were introduced to CaCo-2 cultures and their association with CaCo-2 cells was assessed. Undigested, pepsin-digested and pepsin- and pancreatin-digested alpha-lactalbumin and glycomacropeptide inhibited association. Thus, milk supplemented with alpha-lactalbumin and glycomacropeptide might be effective in inhibiting associations of the pathogens EPEC, Salmonella typhimurium, and Shigella flexneri to intestinal cells.

Kinetic modeling of the generation of 2-and 3-methylbutanal in a heated extract of beef liver

Quantitative control of aroma generation during the Maillard reaction presents great scientific and industrial interest. Although there have been many studies conducted in simplified model systems, the results are difficult to apply to complex food systems, where the presence of other components can have a significant impact. In this work, an aqueous extract of defatted beef liver was chosen as a simplified food matrix for studying the kinetics of the Mallard reaction. Aliquots of the extract were heated under different time and temperature conditions and analyzed for sugars, amino acids, and methylbutanals, which are important Maillard-derived aroma compounds formed in cooked meat. Multiresponse kinetic modeling, based on a simplified mechanistic pathway, gave a good fit with the experimental data, but only when additional steps were introduced to take into account the interactions of glucose and glucose-derived intermediates with protein and other amino compounds. This emphasizes the significant role of the food matrix in controlling the Maillard reaction.

A tunable radial basis function model for nonlinear system identification using particle swarm optimisation

A tunable radial basis function (RBF) network model is proposed for nonlinear system identification using particle swarm optimisation (PSO). At each stage of orthogonal forward regression (OFR) model construction, PSO optimises one RBF unit's centre vector and diagonal covariance matrix by minimising the leave-one-out (LOO) mean square error (MSE). This PSO aided OFR automatically determines how many tunable RBF nodes are sufficient for modelling. Compared with the-state-of-the-art local regularisation assisted orthogonal least squares algorithm based on the LOO MSE criterion for constructing fixed-node RBF network models, the PSO tuned RBF model construction produces more parsimonious RBF models with better generalisation performance and is computationally more efficient.

Nonlinear system identification using particle swarm optimisation tuned radial basis function models

A novel particle swarm optimisation (PSO) tuned radial basis function (RBF) network model is proposed for identification of non-linear systems. At each stage of orthogonal forward regression (OFR) model construction process, PSO is adopted to tune one RBF unit's centre vector and diagonal covariance matrix by minimising the leave-one-out (LOO) mean square error (MSE). This PSO aided OFR automatically determines how many tunable RBF nodes are sufficient for modelling. Compared with the-state-of-the-art local regularisation assisted orthogonal least squares algorithm based on the LOO MSE criterion for constructing fixed-node RBF network models, the PSO tuned RBF model construction produces more parsimonious RBF models with better generalisation performance and is often more efficient in model construction. The effectiveness of the proposed PSO aided OFR algorithm for constructing tunable node RBF models is demonstrated using three real data sets.

Construction of tunable radial basis function networks using orthogonal forward selection

An orthogonal forward selection (OFS) algorithm based on leave-one-out (LOO) criteria is proposed for the construction of radial basis function (RBF) networks with tunable nodes. Each stage of the construction process determines an RBF node, namely, its center vector and diagonal covariance matrix, by minimizing the LOO statistics. For regression application, the LOO criterion is chosen to be the LOO mean-square error, while the LOO misclassification rate is adopted in two-class classification application. This OFS-LOO algorithm is computationally efficient, and it is capable of constructing parsimonious RBF networks that generalize well. Moreover, the proposed algorithm is fully automatic, and the user does not need to specify a termination criterion for the construction process. The effectiveness of the proposed RBF network construction procedure is demonstrated using examples taken from both regression and classification applications.