Mediterranean diet and cancer: epidemiological evidence and mechanism of selected aspects

Background: Populations living in the area of the Mediterranean Sea suffered by decreased incidence of cancer compared with those living in the regions of northern Europe and US countries, attributed to healthier dietary habits. Nowadays, we are assisting to a moving away from the traditional Mediterranean dietary pattern, but whether this changing is influencing risk of cancers is still unclear. The aim of the study was to review recent evidence on potential relationship between the adherence to the Mediterranean diet and cancer. Discussion: The most recent pooled analyses of epidemiological studies supported strongly the hypothesis that the Mediterranean diet may play a role in preventing several types of cancers, especially those of digestive tract, whereas contrasting results were reported for hormone-dependent cancers. Specific aspects of the Mediterranean diet such as high fruit and vegetables and low red processed meat intake may explain such protective effects. Moreover, evidence regarding olive oil and whole grains increase the beneficial effects of such dietary pattern against cancer. Conclusions: Literature evidence actually demonstrates that the increased adherence to the Mediterranean dietary pattern is beneficial to health across populations and may translate a protective effect with certain cancers

One-carbon metabolism and epigenetic programming of mammalian development

One-carbon (1C) metabolism comprises a series of integrated metabolic pathways, including the linked methionine-folate cycles, that provide methyl groups for the synthesis of biomolecules and the epigenetic regulation of gene expression via chromatin methylation. Most of the research investigating the function of 1C metabolism pertains to studies undertaken in the rodent liver. Comparatively little is known about the function of 1C metabolism in reproductive and embryonic cells, particularly in domestic ruminant species. Periconceptional dietary deficiencies in 1C substrates and cofactors are known to lead to epigenetic alterations in DNA methylation in genes that regulate key developmental processes in the embryo. Such modifications can have negative implications on the subsequent development, metabolism and health of offspring. This thesis sought to improve current understanding of the regulation of 1C metabolism in the ruminant liver, ovary and preimplantation embryo through in vivo and in vitro nutritional supplementation experiments coupled with metabolomic, transcriptomic and epigenetic analyses. The first part of this thesis (Chapter 2) assessed the metabolic consequences of dietary methyl deficiency using novel mass spectrometry–based methods that were developed for the quantification of B vitamins, folates and 1C-related amines in sheep liver. This study provided the first comparison of the relative abundance of bioactive 1C metabolites in liver harvested from methyl deficient sheep relative to a control study population of abattoir derived sheep. Relevant reductions in dietary methyl availability led to significant alterations in hepatic 1C metabolite concentrations. Large natural variations in the hepatic concentrations of individual metabolites in both sheep study populations reflected the dietary and genetic variation in our chosen outbred model species. These metabolomics platforms will be useful for investigating 1C metabolism and linked biochemical pathways in order to facilitate future dietary and genetic studies of metabolic health and epigenetic regulation of gene expression. Based on the absence of methionine cycle enzyme transcripts (e.g. MAT1A and BHMT) in the bovine ovary and preimplantation embryo, the second part of this thesis (Chapter 3 and Chapter 4) addressed the hypothesis that ruminant reproductive and embryonic cells are highly sensitive to methyl group availability and, therefore, epigenetic programming during the periconceptional period. Transcript analyses confirmed MAT2A expression in the bovine liver, ovary and at each stage of preimplantation embryo development assessed to Day 8. Transcripts for BHMT isoforms (BHMT and BHMT2) were detected in the bovine ovary but were weak or absent in embryos, highlighting a key difference in methionine metabolism between hepatic and reproductive cells. Bovine embryos were produced in vitro using custom-made media containing 0 (nonphysiological), 10 (low physiological), 50 (high physiological), and 500 µmol/L (supraphysiological) added methionine (Chapter 3). Gross morphological assessments of embryo stage, grade, cell lineage allocation and primary sex ratio revealed that culture in non- and supraphysiological methionine concentrations was detrimental for embryo development, whilst culture in the high physiological concentration appeared to be best. Reduced representation bisulphite sequencing (RRBS) of inner cell mass (ICM) and trophectoderm (TE) cells immunodissected from Day 8 blastocysts demonstrated that culturing embryos in low physiological methionine led to global hypomethylation within both cell lineages. Bioinformatic analyses of differentially methylated genes included gene set enrichment analyses (GSEA). Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that were enriched within the ICM were associated with protein catabolism and autophagy, and significant terms and pathways enriched within the TE were associated with cellular transport. Of particular biological interest was the loss of methylation within regulatory region (DMR2) of the paternally imprinted gene, IGF2R, in the TE following culture in low physiological methionine. Transcript analysis found no significant effect of methionine concentration on the expression of IGF2R or the antisense transcript, AIRN, in the primary cell lineages of the Day 8 bovine preimplantation embryo. Hypomethylation of IGF2R DMR2 has been associated with aberrant IGF2R expression and large offspring syndrome (LOS) in cattle and sheep that were subjected to embryo manipulation during assisted reproductive technology (ART) procedures, such as somatic cell nuclear transfer (SCNT) or non-physiological in vitro embryo culture environments. Chapter 5 sought to evaluate the effect of somatic donor cell type on epigenetic reprogramming via DNA methylation in hepatocytes isolated from cloned sheep. RRBS facilitated the comparison of methylation reprogramming between Finn Dorset (D) clone hepatocytes and their mammary epithelial (OP5) donor cell line; and, Lleyn (L) clone hepatocytes and their Lleyn fetal fibroblast (LFF4) donor cell line. Methylation was most closely correlated between D and L clone hepatocytes than between clones and their respective donor cell lines. In general, hepatocytes were hypomethylated relative to their somatic donor cell nuclei. GSEA identified genes that encoded transcription factor proteins enriched within the ‘Sequence-specific DNA binding’ term (GO:0043565) as differentially methylated between clone hepatocytes and their donor cell lines. In addition, imprinted genes, including IGF2R, were differentially methylated in clone hepatocytes relative to somatic cell nuclei. In summary, this thesis promotes and supports the importance of an optimal methyl balance to support periconceptional development in mammals. The experiments detailed herein provide an insight into the metabolic consequences of dietary methyl deficiency (and excess) in outbred populations of domestic ruminants, with a specific focus on the liver, ovary and preimplantation embryo. The results demonstrate that tissue- and species-specific features of 1C metabolism render ruminant embryonic cells sensitive to methionine inputs within a physiological range. The observation that in vitro embryo culture and manipulation techniques, such as somatic cell nuclear transfer, can cause epigenetic alterations to DNA methylation during preimplantation development provides a basis for further study into the safety and efficacy of emerging assisted reproductive technologies

Health-affecting methyl-donor compounds in sour cherry (prunus cerasus l.) fruit parts in the fruit burgeoning stage

In recent years, numerous studies have confirmed the vital role and therapeutic potential of quaternary ammonium compounds and endogenous formaldehyde in the prevention and treatment of diseases. These compounds participate in the transmethylation processes and play a role in the metabolism and in the regulation of cellular processes. The present research indicates that sour cherry fruit contains large quantities of quaternary ammonium compounds in the early developmental phase (burgeoning). The quantity of methyl-donor compounds (choline, carnitine) and easily mobilizable methyl groups were measured in various fruit parts (stalk, fruit flesh, seed kernel) of five sour cherry cultivars using OPLC technology, and the cultivar dependence of the detected compounds was examined. The results of comparative examinations established the presence of choline in the stalk and seed and of a significant amount of carnitine in the fruit flesh. The clear, significant differences between the genotypes will facilitate the selection of cultivars containing the largest proportion of components beneficial for human health

High prevalence of functional vitamin deficiencies in a psychogeriatric ward

Choline (Ch) is involved in relevant neurochemical processes. It is the precursor and metabolite of acetylcholine (ACh). It plays a role in single-carbon metabolism and is an essential component of different membrane phospholipids (PLs). These PLs are structural components of cell membranes, and involved in intraneuronal signal transduction. An increased ACh release was found after Ch treatment in rat corpus striatum slices. An in vivo proton magnetic resonance study has analyzed Ch ingestion effect. This work which represents the first non invasive study for exploring in vivo human brain neurochemistry showed the transfer of an oral Ch load in the brain of normal volunteers. These results were not confirmed by other in vivo studies. Cellular membranes breakdown is suggested as a feature of neurodegeneration in acute (stroke) and chronic (Alzheimer’s and vascular dementias) brain disorders. The effects of exogenous CCPLs on different brain areas were largely studied. Our group has assessed the influence of treatment with the CCPL, choline alphoscerate (GPC) on brain cholinergic neurotransmission markers in an animal model of brain vascular injury. A neuroprotective effect of GPC alone or in association with acetylcholinesterase inhibitor, galantamine was found. These results suggest that GPC could stimulate the expression of vesicular ACh transporter and Ch transporter primarily in areas involved in cognitive processes. These cholinergic markers could represent an appropriate mean to investigate brain cholinergic pathways. In the lack of novel therapeutic strategies, safe compounds developed since a long time such as the CCPLs could have still a place in pharmacotherapy and would merit to be investigated by new clinical studies