European Journal of Nutrition (v.51, #7)

Experimental evidence indicates a strong connection between oxidative damage, cancer, and aging. Epidemiological observations suggest that a diet rich in fruits and vegetables is associated with lower incidence of some cancers and longer life expectancy; since fruits and vegetables contain natural antioxidants, a considerable effort has been dedicated to understanding their effects in experimental studies and in human trials.A: Effects of antioxidant-containing food and supplements on oxidation damage in humans. Intervention trials employing a variety of biomarkers have shown either a slight decrease in oxidation damage or no effect. B: Effects of selected antioxidants on mortality and cancer incidence. β-carotene and α-tocopherol, alone or in combination, increase cardiovascular and all-cause mortality or have no effect. In some studies, β-carotene and retinyl palmitate significantly increase the progression of lung cancer and aggressive prostate cancer. Protection against cardiovascular mortality or no effect of vitamin E has been reported, with an increase of all-cause mortality at dosages greater than 150 IU/day. Selenium showed beneficial effects on gastrointestinal cancer and reduced the risk of lung cancer in populations with lower selenium status. For multivitamin and mineral supplementation, no significant reduction of mortality or cancer incidence was observed, but some reports indicate a possible preventive effect in cervical cancer.The majority of supplementation studies indicate no variation of general mortality and of cancer incidence or a detrimental effect on both. Antioxidant supplements so far tested seem to offer no improvement over a well-balanced diet, possibly because of the choice of the substances tested or of an excessive dosage. However, new natural or synthetic compounds effective in vitro and in experimental studies might still be worth investigating in human trials.
Keywords: Free radicals; Antioxidants; Aging; Cancer

Assessment of iron absorption in mice by ICP-MS measurements of 57Fe levels by Veronica Fiorito; Simonetta Geninatti Crich; Lorenzo Silengo; Fiorella Altruda; Silvio Aime; Emanuela Tolosano (783-789).
The study of iron metabolism is essential in nutritional sciences as iron deficiency is one of the most common nutritional deficiencies in humans and represents a serious health problem worldwide. The mouse is utilized as a unique and powerful model for the identification and characterization of genes involved in iron metabolism and for studying the pathogenesis of iron disorders. Thus, sophisticated and sensitive techniques have been developed to study iron metabolism in this animal model. In particular, iron absorption has been studied in mice by using the radioisotopes 55Fe and 59Fe in tied-off or dissected and everted duodenal segments. Nevertheless, several drawbacks discourage the extended use of these approaches.Here, we report the use of the stable isotope 57Fe to measure iron absorption in mice. We show that after oral administration of 57Fe-containing solutions, it is possible to measure both duodenal iron retention and duodenal iron transfer to specific organs, using inductively coupled plasma mass spectrometry (ICP-MS). As 57Fe is administered orally, no surgical operation is needed before the end of the experiment, thus allowing the measurement of iron absorption under physiologic conditions. Moreover, the use of ICP-MS for 57Fe detection ensures high sensitivity and provides quantitative data. Finally, the use of a stable isotope enables the measurement of both iron absorption and histologic and/or biochemical analyses in the same animal.The use of 57Fe to measure iron absorption in mice, therefore, represents an alternative to radioisotope-based methods, providing a new tool to extend our knowledge on the mechanism of iron absorption.
Keywords: Iron absorption; Mice; ICP-MS

Coenzyme Q10 supplementation ameliorates inflammatory signaling and oxidative stress associated with strenuous exercise by Javier Díaz-Castro; Rafael Guisado; Naroa Kajarabille; Carmen García; Isabel M. Guisado; Carlos de Teresa; Julio J. Ochoa (791-799).
Exhausting exercise induces muscle damage associated with high production of free radicals and pro-inflammatory mediators.The objective of this study was to determine for the first time and simultaneously whether oral coenzyme Q10 (CoQ10) supplementation can prevent over-expression of inflammatory mediators and oxidative stress associated with strenuous exercise.The participants were classified in two groups: CoQ10 group (CG) and placebo group (PG). The physical test consisted in a constant run (50 km) that combined several degrees of high effort (mountain run and ultra-endurance), in permanent climbing.Exercise was associated with an increase in TNF-α, IL-6, 8-hydroxy-2′-deoxyguanosine (8-OHdG), and isoprostane levels, revealing the degree of inflammation and oxidative stress induced. Oral supplementation of CoQ10 during exercise was efficient reducing oxidative stress (decreased membrane hydroperoxides, 8-OHdG and isoprostanes generation, increased catalase, and total antioxidant status), which would lead to the maintenance of the cell integrity. Data obtained also indicate that CoQ10 prevents over-expression of TNF-α after exercise, together with an increase in sTNF-RII that limits the pro-inflammatory actions of TNF. Moreover, CoQ10 supplementation reduced creatinine production.CoQ10 supplementation before strenuous exercise decreases the oxidative stress and modulates the inflammatory signaling, reducing the subsequent muscle damage.
Keywords: High-intensity (strenuous) exercise; Coenzyme Q10 ; Oxidative damage; Inflammation

Coffee does not modify postprandial glycaemic and insulinaemic responses induced by carbohydrates by Katja A. Hätönen; Jarmo Virtamo; Johan G. Eriksson; Harri K. Sinkko; Iris Erlund; Pekka Jousilahti; Jaana M. Leiviskä; Liisa M. Valsta (801-806).
Strong epidemiological evidence suggests that coffee consumption is associated with lower risk of type 2 diabetes. In postprandial studies, however, caffeine consumption has been associated with impaired glucose regulation.To study the acute effects of coffee and caffeine-containing soft drinks on glycaemic and insulinaemic responses.Twelve healthy volunteers were served each test food once and the reference glucose solution twice, containing 50 g of available carbohydrates, after an overnight fast at 1-week intervals in a random order. Capillary blood samples were drawn at 15–30 min intervals for 2 h after each study meal. The incremental areas under the curve (IAUC), glycaemic index (GI) and insulinaemic index (II), were calculated to estimate the glycaemic and insulinaemic responses.Glucose and insulin responses of coffees with glucose containing 150 or 300 mg of caffeine did not differ from responses of pure glucose solution; the GIs were 104 and 103, and the IIs were 89 and 92, respectively. When a bun or sucrose and milk were consumed together with coffee, lower GI values and insulin responses were observed, reflecting the carbohydrate quality and protein content of the accompaniments. Sucrose-sweetened cola produced a high GI value of 90 and an II of 61.Coffee does not modify glycaemic and insulinaemic responses when ingested with a carbohydrate source. Therefore, there is no need to avoid coffee as a choice of beverage in GI testing.
Keywords: Coffee; Glucose response; Glycemic index; Insulin response; Insulinemic index

Moderate physical training attenuates muscle-specific effects on fibre type composition in adult rats submitted to a perinatal maternal low-protein diet by Carol Góis Leandro; Wellington da Silva Ribeiro; José Antônio dos Santos; Adriano Bento-Santos; Carlos Henrique Lima-Coelho; Filippe Falcão-Tebas; Cláudia Jacques Lagranha; Sandra Lopes-de-Souza; Raul Manhães-de-Castro; Ana Elisa Toscano (807-815).
To verify whether moderate physical training affects the muscle fibre composition of adult rats subjected to a low protein diet during the perinatal period.Male Wistar rats were divided into two groups according to their mother’s diet during gestation and lactation: control (17% casein, C) and low-protein (8% casein, LP). On postnatal day 60, half of each group was submitted to moderate physical training (8 weeks, 5 days/week−1, 60 min/day−1, at 70% of VO2max, T) or not. After the physical training period, soleus and extensor digitorum longus (EDL) muscles were removed. Myofibrillar ATPase staining was used to classify muscle fibres as type I, IIa, IIb, and intermediate.In the EDL muscle, LP rats showed no changes in the fibre type proportion. Both the C + T and LP + T groups showed a higher percentage of fibres of type IIa, and a lower proportion of fibres of type IIb. In the soleus muscle, LP animals showed a reduction in the proportion of fibre types I and intermediate. C + T rats showed an increase in the fibre type I and IIa. In the LP + T rats, the proportions of the fibre types remained similar to control rats.Moderate physical training acts as a positive environmental stimulus that reverts the effects of a perinatal low-protein diet on the proportion of fibre types in skeletal muscle.
Keywords: Perinatal undernutrition; Physical exercise; Programming; Muscle type fibre; Rats

Diallyl trisulfide-induced prostate cancer cell death is associated with Akt/PKB dephosphorylation mediated by P-p66shc by Andzelika Borkowska; Alicja Sielicka-Dudzin; Anna Herman-Antosiewicz; Michal Wozniak; Donatella Fedeli; Giancarlo Falcioni; Jedrzej Antosiewicz (817-825).
P66Shc, an isoform of adaptor proteins, is known to mediate various signals including those leading to apoptosis or cell proliferation. Previously, we have shown that diallyl trisulfide (DATS)-induced prostate cancer cell death was mediated by increased ROS formation. In this study, we investigated the role of p66Shc protein and its serine 36 phosphorylation in DATS induced decrease in prostate cancer cell viability (PC-3).PC-3 prostate cancer cells were used in this study. Stable cell lines expressing p66ShcS36A or an empty vector have been obtained. Cell viability, concentration of ROS, changes in P-p66Shc and P-Akt and DNA damage were determined.We observed that DATS treatment increased p66Shc phosphorylation at serine 36. Importantly, the phosphorylation was abolished by JNK inhibitor SP600125. Cells expressing plasmid-encoded variant of p66ShcS36A showed much higher resistance to DATS-induced cells death. In addition to that, we observed that DATS-induced ROS formation was completely abolished in cells expressing the p66ShcS36A variant. Interestingly, SP600125 proved to prevent DATS-induced Akt inactivation. In order to confirm that the observed effect is related to phosphorylation of p66Shc, we performed experiments on a stable cell line expressing p66ShcS36A. In such cells, DATS-induced Akt dephosphorylation was significantly reduced. On the other hand, hydrogen peroxide induced Akt activation in PC-3 cells, which was abrogated in cells expressing p66ShcS36A.Our results uncover a novel signaling pathway with p66Shc being indispensable for DATS-induced inactivation of Akt due to hypophosphorylation.
Keywords: Garlic; Oxidative stress; c-jun kinase; Stress

Chemopreventive effects of in vitro digested and fermented bread in human colon cells by Wiebke Schlörmann; Beate Hiller; Franziska Jahns; Romy Zöger; Isabell Hennemeier; Anne Wilhelm; Meinolf G. Lindhauer; Michael Glei (827-839).
Bread as a staple food product represents an important source for dietary fibre consumption. Effects of wheat bread, wholemeal wheat bread and wholemeal rye bread on mechanisms which could have impact on chemoprevention were analysed in colon cells after in vitro fermentation.Effects of fermented bread samples on gene expression, glutathione S-transferase activity and glutathione content, differentiation, growth and apoptosis were investigated using the human colon adenoma cell line LT97. Additionally, apoptosis was studied in normal and tumour colon tissue by determination of caspase activities.The expression of 76 genes (biotransformation, differentiation, apoptosis) was significantly upregulated (1.5-fold) in LT97 cells. The fermented bread samples were able to significantly increase glutathione S-transferase activity (1.8-fold) and glutathione content (1.4-fold) of the cells. Alkaline phosphatase activity as a marker of differentiation was also significantly enhanced (1.7-fold). The fermented bread samples significantly inhibited LT97 cell growth and increased the level of apoptotic cells (1.8-fold). Only marginal effects on apoptosis in tumour compared to normal tissue were observed.This is the first study which presents chemopreventive effects of different breads after in vitro fermentation. In spite of differences in composition, the results were comparable between the bread types. Nevertheless, they indicate a potential involvement of this staple food product regarding the prevention of colon cancer.
Keywords: Apoptosis; Bread; Colon cancer; Dietary fibre; In vitro fermentation

Moderate effects of apple juice consumption on obesity-related markers in obese men: impact of diet–gene interaction on body fat content by Stephan W. Barth; Tatiana C. L. Koch; Bernhard Watzl; Helmut Dietrich; Frank Will; Achim Bub (841-850).
The effect of polyphenol-rich cloudy apple juice (CloA) consumption on plasma parameters related to the obesity phenotype and potential effects of interactions between CloA and allelic variants in obesity candidate genes were assessed in obese men.In this controlled, randomized, and parallel study, n = 68, non-smoking, non-diabetic men with a BMI ≥27 kg/m2 received 750 mL/day CloA (802.5 mg polyphenols) or 750 mL/day control beverage (CB, isocaloric equivalent to CloA) for 4 weeks. Further, study participants were genotyped for single-nucleotide polymorphisms in PPARγ (rs1801282), UCP3 (rs1800849), IL-6 (rs1800795), FABP2 (rs1799883), INSIG2 (rs7566605), and PGC1 (rs8192678) genes. At the beginning and at the end of intervention plasma lipids, distinct adipokines and cytokines as well as anthropometric parameters were determined.CloA compared to CB had no significant effect on plasma lipids, plasma adipokine and cytokine levels, BMI, and waist circumference. However, CloA consumption significantly reduced percent body fat compared to CB (∆ % body fat: CloA: −1.0 ± 1.3 vs. CB: −0.2 ± 0.9, p < 0.05). The IL-6-174 G/C polymorphism showed an interaction with body fat reduction induced by CloA. Solely in C/C, but not in G/C or G/G variants, a significant reduction in body fat after 4 weeks of CloA intervention was detectable.The observed diet–gene interaction might be a first indication for the impact of individual genetic background on CloA-mediated bioactivity on obesity-associated comorbidities.
Keywords: Polyphenols; Human intervention study; Inflammation; Adipokines; Cytokines

Plasma ochratoxin A levels, food consumption, and risk biomarkers of a representative sample of men and women from the Molise region in Italy by Romina di Giuseppe; Terenzio Bertuzzi; Filippo Rossi; Silvia Rastelli; Annalisa Mulazzi; Jessica Capraro; Amalia de Curtis; Licia Iacoviello; Amedeo Pietri (851-860).
Ochratoxin A (OTA) is a mycotoxin present in food that can be found in human blood, due to its long half-life. Plasma OTA detection represents a good parameter for evaluating the exposure at the population level.The relation between plasma OTA levels, dietary habits, and specific disease risk biomarkers (body mass index (BMI), C-reactive protein (CRP), and cardiovascular risk score) was investigated.The study involved 327 subjects (150 men and 177 women) aged between 38 and 48 years. Food consumption was evaluated by means of the EPIC questionnaire; plasma OTA was measured by HPLC; CRP was determined in fresh serum samples by a latex particle-enhanced immunoturbidimetric assay.OTA was detected in 99.1% of plasma samples (LOD 25 ng/L); the mean ± SD value was 0.229 ± 0.238 ng/mL. However, only 5.2% of samples exceeded 500 ng/L, considered the threshold for a possible pathogenic activity. The estimated mean daily dietary intake of OTA resulted 0.452 ± 0.468 ng/kg body weight (bw)/day, markedly lower than the tolerable daily intake set by EFSA (17.1 ng/kg bw/day). Processed and mutton/lamb meat were found to contribute most to plasma OTA variance. Nevertheless, cereals, wine, beer, and jam/honey consumption correlated positively with OTA levels. Plasma OTA showed a significant positive association with CRP and cardiovascular risk score (β = 0.20 ± 0.08; P = 0.015 and β = 0.25 ± 0.08; P = 0.001, respectively); however, the association was present in men but not in women.Even if the hypothesis of a possible hepatic toxicity of OTA in humans is yet to be verified, the positive association between plasma OTA and CRP may indicate a possible role of OTA in inflammation status and consequently in the genesis of cardiovascular diseases and cancer.
Keywords: Ochratoxin A; C-reactive protein; Cardiovascular disease; Cancer

Estrogen modulates abdominal adiposity and protects female mice from obesity and impaired glucose tolerance by Renee E. Stubbins; Valerie B. Holcomb; Jina Hong; Nomelí P. Núñez (861-870).
Obesity increases the risk of diabetes. The dysregulation of estrogen metabolism has been associated with the susceptibility to obesity and diabetes. Here, we explore the role estrogen plays in sex differences in obesity and glucose metabolism, specifically adipocyte biology.We randomized C57BL/6 J male, non-ovariectomized female, ovariectomized female, and ovariectomized female mice supplemented with 17β estradiol to receive a calorie-restricted, low- or a high-fat diet (15 mice per group). We measured weight gained, calories consumed, percent body fat, abdominal adipose tissue, adipocyte size, lipogenic and adipogenic gene expression, and glucose tolerance.Male mice had a higher susceptibility to obesity than intact female mice. However, removal of the ovaries in female mice eliminated the protection to obesity and estrogen supplementation restored this protection. Male and ovariectomized female mice gained weight predominately in the form of abdominal adipose tissue possibly due to an increase in adipocyte size. Moreover, for mice consuming the high-fat diet, male and ovariectomized female mice had significantly higher levels of leptin mRNA and lower hormone-sensitive lipase mRNA relative to intact female mice and ovariectomized female mice supplemented with estrogen. Additionally, estrogen had a strong inhibitory effect on key adipogenic genes in non-ovariectomized female and ovx-female mice supplemented with estrogen. Finally, we show that male and ovariectomized female mice consuming the high-fat diet had a higher incidence of glucose intolerance.Estrogen protects female mice from obesity and impaired glucose tolerance possibly by modulating the expression of genes regulating adipogenesis, lipogenesis, and lipolysis.
Keywords: Obesity; Sex; Ovariectomy; Diet; Calorie-restriction

Influence of bread crust-derived Maillard reaction products on phosphorus balance in rats by Irene Roncero-Ramos; Cristina Delgado-Andrade; Rebeca Alonso-Olalla; María Pilar Navarro (871-879).
Maillard reaction products (MRP) improve food palatability and are linked to some positive biological actions. However, diverse negative consequences, some related to protein damage and mineral availability, have been established.We investigated the effects of MRP, from a bread crust diet, on phosphorus bioavailability and tissue distribution in rats to determine whether these effects are related to the molecular weight of browning products.During a study period of 88 days, rats were fed either a control diet or one of the following: with bread crust as a source of MRP, or one with its soluble high molecular weight, soluble low molecular weight or insoluble fraction (bread crust, HMW, LMW and insoluble diets, respectively). In the final week, a phosphorus balance was performed, after which the animals were sacrificed and some organs removed to analyse phosphorus content. A second balance was carried out throughout the experimental period to calculate phosphorus retention.Phosphorus balance in the last week was unchanged. However, considering the whole experimental period, a trend towards improved bioavailability, significant in the HMW group, was observed. Higher phosphorus concentrations were measured in the small intestine and bone.The consumption of MRP derived from bread did not alter phosphorus retention, due to increased bioavailability, especially concerning HMW compounds. The overall phosphorus body content remained unchanged and there were no changes in the bone, its principal metabolic destination. However, MRP consumption markedly raised phosphorus levels at the digestive level, especially when consumed as isolate fractions. The slower rate of stomach emptying is assumed to be related to this effect.
Keywords: Maillard reaction products; Phosphorus bioavailability; Tissue distribution; Bone

Procyanidin B2 (PB2) is a naturally occurring flavonoid widely found in cocoa, red wine and grape juice. Recent studies have suggested that PB2 could protect against oxidative stress- and chemical-induced injury in colonic cells by modulating the endogenous cellular defence. However, the precise mechanism for this protection is not fully understood. Herein, we examined the effect of PB2 on the expression of one of the major antioxidant/detoxificant enzymes related to intestinal protection, the glutathione S-transferase P1 (GSTP1), and the molecular mechanisms involved.Human colonic Caco-2 cells were treated with PB2 at different times and enzymatic activity, and mRNA and protein levels of GSTP1 were evaluated. The nuclear translocation of the transcription factor NF-erythroid 2-related factor (Nrf2) and the phosphorylation states of specific proteins central to intracellular signalling cascades were also investigated.PB2 induced the expression and activity of GSTP1 and the nuclear translocation of Nrf2. Interestingly, two important signalling proteins involved in Nrf2 translocation, the extracellular signal-regulated protein kinases (ERKs) and the p38 mitogen-activated protein kinase (MAPK) were also activated. Further experiments with specific inhibitors of both pathways confirmed their critical role in the beneficial effects induced by PB2.The present results show that PB2 protects against oxidative injury in colonic cells and up-regulate the expression of GSTP1 via a mechanism that involves ERK and p38 MAPK activation and Nrf2 translocation. These results provide a molecular basis for the potential contribution of PB2 in the prevention of oxidative stress-related intestinal injury and gut pathologies.
Keywords: Cocoa flavonoids; Glutathione enzymes; Signalling pathways; Oxidative stress

4-Hydroxyisoleucine stimulates glucose uptake by increasing surface GLUT4 level in skeletal muscle cells via phosphatidylinositol-3-kinase-dependent pathway by Natasha Jaiswal; Chandan K. Maurya; K. Venkateswarlu; P. Sukanya; Arvind K. Srivastava; Tadigoppula Narender; Akhilesh K. Tamrakar (893-898).
To determine the effect of 4-Hydroxyisoleucine (4-HIL), an unusual amino acid isolated from the seeds of Trigonella foenum-graecum, on glucose uptake and the translocation of glucose transporter 4 (GLUT4) to plasma membrane in skeletal muscle cells and to investigate the underlying mechanisms of action.Rat skeletal muscle cells (L6-GLUT4myc) were treated with 4-HIL, and the effect on glucose uptake was determined by measuring the incorporation of radio-labeled 2-deoxy-[3H]-d-glucose (2-DG) into the cell. Translocation of GLUT4myc to plasma membrane was measured by an antibody-coupled colorimetric assay.The prolonged exposure (16 h) of L6-GLUT4myc myotubes to 4-HIL caused a substantial increase in the 2-DG uptake and GLUT4 translocation to the cell surface, without changing the total amount of GLUT4 and GLUT1. Cycloheximide treatment reversed the effect of 4-HIL on GLUT4 translocation to the basal level suggesting the requirement of new protein synthesis. The 4-HIL-induced increase in GLUT4 translocation was completely abolished by wortmannin, and 4-HIL significantly increased the basal phosphorylation of AKT (Ser-473), but did not change the mRNA expression of AKT, IRS-1, GLUT4, and GSK3β.Results suggest that 4-HIL stimulates glucose uptake in L6-GLUT4myc myotubes by enhancing translocation of GLUT4 to the cell surface in a PI-3-kinase/AKT-dependent mechanism.
Keywords: Insulin resistance; 4-Hydroxyisoleucine; GLUT4 translocation; Skeletal muscle