François Laporte

Mediterranean diet and the French paradox: Two distinct biogeographic concepts for one consolidated scientific theory on the role of nutrition in coronary heart disease

Time for primary review 26 days. Scientists and physicians have long been debating the Mediterranean-style diet and the French paradox for coronary heart disease (CHD). However, folksy they sound, these two biogeographic concepts can still be very useful to explain unexpected or controversial medical and scientific data, such as the low mortality rate from CHD in Mediterranean Southern Europe and in France as compared with other European countries. Understanding these concepts may help improve our ability to treat and prevent CHD. Most of the present confusion probably comes from the consistent underestimation by physicians and scientists of the role of nutrition in CHD. This article is not aimed at giving a comprehensive review of these two complex notions, which have to be analyzed in a broad geographic, climatic, agricultural, historical and socioeconomic context. We will only provide a superficial overview, in relation to the epidemiology of CHD. Finally, we will try to introduce the two concepts as a fundamental premise of a new scientific theory on the role of nutrition in CHD, a theory that remains to be fully formulated. ### 2.1 Definition The definition of the Mediterranean-style diet varies according to the particular Mediterranean area that is considered. For instance, the Greek version of the Mediterranean diet is dominated by the consumption of olive oil and by a high consumption of vegetables and fruits [1]. Since antioxidants are common in these foods, an antioxidant action may provide a plausible explanation for the apparent benefits of that diet [1]. According to another version, however, the Mediterranean diet is a non-strict vegetarian diet rich in oleic acid, omega-3 fatty acids, fiber, vitamins of the B group and various antioxidants, but low in saturated and polyunsaturated fat [2]. With that wider definition, the expected benefits for the prevention of CHD go far … * Corresponding author. Laboratoire du Stress Cardiovasculaire et Pathologies Associees, UFR de Medecine et Pharmacie, Domaine de la Merci, 38706 La Tronche, Grenoble, France. Tel.: +33-4-7663-7471; fax: +33-4-7663-7152

The mycotoxin Zearalenone induces apoptosis in human hepatocytes (HepG2) via p53-dependent mitochondrial signaling pathway

Zearalenone (Zen) is a fusarial mycotoxin commonly found in several food commodities worldwide. It is frequently implicated in reproductive disorders and exerts several genotoxic effects in vivo and in vitro. In response to DNA damage, cells may undergo an intricate network of different pathways including apoptosis. Meanwhile, data regarding the induction of apoptosis after Zen exposure are limited. Thus, the aim of this study was to demonstrate whether Zen-induced DNA damage can lead to apoptosis as a stress response and which pathways are undertaken. Our results clearly show that Zen reduces cell proliferation in HepG2 cells in a dose-dependent manner as attested by the MTT assay (IC50%, 100microM). The analysis of propidum iodide uptake has shown that the amount of necrotic cells was about 6% among 55% of dead cells (at 120microM of Zen). The involvement of apoptosis as a major cause of Zen-induced cell death was further confirmed but results of caspase-3 activity showed a Zen-dose dependant increase. Furthermore, results of microarrays analysis have shown that Zen induced an upregulation of ATM and p53 genes family. ATM pathway responds primarily to DNA double-strand breaks and has been involved in the activation and stabilization of p53. The activation of p53 was accompanied by an upregulation of GADD45 to arrest the cell cycle and to allow the repair mechanisms to take place. In addition, results of genes profiling as well as western-blotting analysis showed that Zen increased the ratio of pro-apoptotic factors/anti-apoptotic factors which led to the loss of mitochondrial potential, Bax translocation and cytochrome c release. Once released, cytochome c activates caspase 9 which in turn activates caspase-3 and enhances apoptosis. In summary, these data suggested that Zen induced apoptosis in a dose-dependent manner in HepG2 cells via a p53-dependent mitochondrial pathway.

Dietary Flavonoids Increase Plasma Very Long-Chain (n-3) Fatty Acids in Rats

Flavonoids probably contribute to the health benefits associated with the consumption of fruit and vegetables. However, the mechanisms by which they exert their effects are not fully elucidated. PUFA of the (n-3) series also have health benefits. Epidemiological and clinical studies have suggested that wine flavonoids may interact with the metabolism of (n-3) PUFA and increase their blood and cell levels. The present studies in rats were designed to assess whether flavonoids actually increase plasma levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the main very long-chain (n-3) PUFA. Rats were fed a corn-derived anthocyanin (ACN)-rich (ACN-rich) or ACN-free diet with constant intakes of plant and marine (n-3) PUFA for 8 wk (Expt. 1). Plasma fatty acids were measured by GC. The ACN-rich diet contained ~0.24 ± 0.01 mg of ACN/g pellets. There were no significant differences between groups in the main saturated, monounsaturated, and (n-6) fatty acids. In contrast, plasma EPA and DHA were greater in the ACN-rich diet group than in the ACN-free diet group (P < 0.05). We obtained similar results in 2 subsequent experiments in which rats were administered palm oil (80 μL/d) and consumed the ACN-rich or ACN-free diet (Expt. 2) or were supplemented with fish oil (60 mg/d, providing 35 mg DHA and 12 mg EPA) and consumed the ACN-rich or ACN-free diet (Expt. 3). In both experiments, plasma EPA and DHA were significantly greater in the ACN-rich diet group. These studies demonstrate that the consumption of flavonoids increases plasma very long-chain (n-3) PUFA levels. These data confirm previous clinical and epidemiological studies and provide new insights into the health benefits of flavonoids.

Protective effect of dietary n-3 polyunsaturated fatty acids on myocardial resistance to ischemia-reperfusion injury in rats

Dietary n-3 polyunsaturated fatty acids (PUFA) reduce coronary heart disease (CHD) complications, such as chronic arrhythmia and sudden cardiac death. Improved myocardial resistance to ischemia-reperfusion injury results in smaller myocardial infarction, which is a major factor in the occurrence of CHD complications. We hypothesized that a specific dietary fatty acid profile (low in saturated and n-6 PUFA but high in plant and marine n-3 PUFA) may improve myocardial resistance to ischemia-reperfusion injury and reduce infarct size. To test this assumption, we used a well-defined rat model of myocardial infarction. Based on our results, in comparison to a diet that is high in either saturated or n-6 PUFA but poor in plant and marine n-3 PUFA, a diet that is low in saturated fats and n-6 PUFA but rich in plant and marine n-3 PUFA results in smaller myocardial infarct size (P < .01). The effects of the 3 diets were also examined by analyzing the fatty acid composition of plasma, erythrocyte cell membranes, and the phospholipids of myocardial mitochondria. The results show a great accumulation of n-3 PUFA and a parallel decrease in arachidonic acid, the main n-6 PUFA, in plasma, cell membranes, and cardiac mitochondria (P < .0001). We conclude that improved myocardial resistance to ischemia-reperfusion may be one of the critical factors explaining the protective effects of dietary n-3 PUFA against CHD complications in humans. In addition to increasing n-3 PUFA intake, an optimal dietary pattern aimed at reducing cardiovascular mortality should include a reduction of the intake of both saturated and n-6 PUFA.

Quantitative determination of plasma vitamin K1 by high-performance liquid chromatography coupled to isotope dilution tandem mass spectrometry

Plasma vitamin K1 (phylloquinone) determination is commonly used for the diagnosis of vitamin K deficiency in patients suffering from lipid malabsorption. Moreover, current evidence that adequate vitamin K intake, and correspondingly adequate plasma vitamin K1 concentration, could also be of importance in relation to bone and brain diseases emphasizes the need to improve the current analytical methods. We developed a liquid chromatography coupled to tandem mass spectrometry method using a stable isotope ring-D4-labeled internal standard of vitamin K1 and operating in the multiple reaction monitoring mode by the selection of a precursor and product ions. The atmospheric pressure chemical ionization (APCI) method was shown to be more sensitive than electrospray ionization. After a single-step extraction with cyclohexane, chromatographic separation was performed on a C18 column with an isocratic mobile phase. The linearity was up to 5400ng/L, and the limit of detection was 14ng/L. Intra- and interrun precision were 2.4% and 8.3%, respectively, for the lower limit of the reference range. Recovery was better than 98%. The method is simple and reliable, allowing accurate vitamin K1 measurement in plasma samples from healthy subjects and patients suffering from vitamin K deficiency.

Zinc, copper and antioxidant enzyme activities in healthy elderly Tunisian subjects

Trace elements like zinc and copper play an important role in maintaining metabolic homeostasis in elderly subjects and is therefore expected to have a crucial effect on antioxidant mechanism. The objective of the present study was to investigate age-related variations of zinc, copper and antioxidant enzyme activities (superoxide dismutase: SOD, glutathione peroxidase: GPx and catalase: CAT) taking into account gender differences in a Tunisian elderly population. A group of 100 healthy elderly subjects (55-85 years old) were then separated in three sub-groups according to age intervals. A control group of 100 adults aged between 30 and 45 years was considered. The obtained results confirmed the decrease of plasma zinc level with age increase in both men and women. Moreover, prevalence of zinc deficiency increased with age: normal zinc concentration was obtained in about 60% of adults and only in 35% of the elderly subjects over 75 years old. No significant variation was obtained for copper concentration. GPx and SOD activities were lower in aged subjects in comparison to adults. Zinc and antioxidant enzyme activities were found to be negatively correlated to age. However, an investigation on a large size sample with various health and well-controlled dietary statuses should be conducted for a better understanding of the zinc or copper metabolism and their effect on oxidant stress during aging.

Influence of dietary habits, age and gender on plasma fatty acids levels in a population of healthy Tunisian subjects

The fatty acids composition of circulating blood lipids is expected to be altered by many factors (ageing, dietary intake, lifestyle...). In addition to the ageing consequences on their lipid status, elderly subjects represent a population at risk of nutritional imbalance. The main objective of the present study was to investigate the associations between dietary habits and the plasma fatty acids patterns in a healthy Tunisian population with an emphasis on the gender and ageing differences for the 6-desaturase activity and the EFA proportions. Nutritional habits and plasma fatty acids compositions have been therefore evaluated in 200 healthy volunteers (104 women and 96 men) aged between 40 and 82years old. The findings revealed that the 6-desaturase activity was reduced in elderly subjects (by 24% and 10% in women and men respectively). Moreover, DHA (C22:6n-3) and AA (C20:4n-6) were found to increase respectively in high fish and meat consumers. Plasma fatty acids composition could be sensitive to dietary habits according to particular food items and should then help for the establishment of optimal nutritional proportions.

Antimutagenic, antigenotoxic and antioxidant activities of Acacia salicina extracts (ASE) and modulation of cell gene expression by H2O2 and ASE treatment.

The total oligomers flavonoids (TOF), chloroform, petroleum ether and aqueous extracts from Acacia salicina, were investigated for the antioxidative, cytotoxic, antimutagenic and antigenotoxic activities. The viability of K562 cells were affected by all extracts after 48 h exposure. Our results showed that A. salicina extracts have antigenotoxic and/or antimutagenic activities. TOF and chloroform extracts exhibit antioxidant properties, expressed by the capacity of these extracts to inhibit xanthine oxidase activity. To further explore the mechanism of action of A. salicina extracts, we characterized expression profiles of genes involved in antioxidant protection and DNA repair in the human lymphoblastic cell line K562 exposed to H2O2. Transcription of several genes related to the thioredoxin antioxidant system and to the DNA base-excision repair pathway was up-regulated after incubation with chloroform, TOF and petroleum ether extracts. Moreover genes involved in the nucleotide-excision repair pathway and genes coding for catalase and Mn-superoxide-dismutase, two important antioxidant enzymes, were induced after incubation with the chloroform extract. Taken together, these observations provide evidence that the chloroform and TOF extracts of A. salicina leaves contain bioactive compounds that are able to protect cells against the consequences of an oxidative stress.

Cellular and molecular mechanisms activating the cell death processes by chalcones: Critical structural effects

Chalcones are naturally occurring compounds with diverse pharmacological activities. Chalcones derive from the common structure: 1,3-diphenylpropenone. The present study aims to better understand the mechanistic pathways triggering chalcones anticancer effects and providing evidences that minor structural difference could lead to important difference in mechanistic effect. We selected two recently investigated chalcones (A and B) and investigated them on glioblastoma cell lines. It was found that chalcone A induced an apoptotic process (type I PCD), via the activation of caspase-3, -8 and -9. Chalcone A also increased CDK1/cyclin B ratios and decreased the mitochondrial transmembrane potential (ΔΨm). Chalcone B induced an autophagic cell death process (type II PCD), ROS-related but independent of both caspases and protein synthesis. Both chalcones increased Bax/Bcl2 ratios and decreased Ki67 and CD71 antigen expressions. The present investigation reveals that despite the close structure of chalcones A and B, significant differences in mechanism of effect were found.

Antigenotoxic and antioxidant activities of isorhamnetin 3-O neohesperidoside from Acacia salicina

Antioxidant activity of isorhamnetin 3-O neohesperidoside (I3ON), isolated from the leaves of Acacia salicina, was determined by the ability of this compound to inhibit lipid peroxidation and to protect against hydroxyl radical–induced DNA damage in pKS plasmid DNA and Escherichia coli cultures. Antigenotoxic activity was assessed by using the comet assay. The IC50 value of the inhibitory activity toward lipid peroxidation by I3ON is 0.6 mM. This compound was also able to protect against hydroxyl radical–induced DNA damage in pKS plasmid DNA. Moreover, this compound induced an inhibitory activity toward H2O2-induced genotoxicity. The protective effect exhibited by this molecule was also determined by analysis of gene expression as a response to an oxidative stress, using a cDNA microarray. Transcription of several genes related to the antioxidant system (HMOX2 and TXNL) and to the DNA repair pathway (XPC, POLD1, POLD2, PCNA, DDIT3, APEX, and LIG4) were upregulated after incubation with I3ON. Taken together, these observations provide evidence that the I3ON, isolated from the leaves of A. salicina, is able to protect cells against oxidative stress.