Erik J.B. Ruijters

Epigenetics: prenatal exposure to genistein leaves a permanent signature on the hematopoietic lineage

Recent studies demonstrate that maternal diet during pregnancy results in long‐lasting effects on the progeny. Supplementation of maternal diet with genistein, a phytoestrogen ubiquitous in the daily diet, altered coat color of agouti mice due to epigenetic changes. We studied hematopoiesis of mice prenatally exposed to genistein (270 mg/kg feed) compared with that of mice prenatally exposed to phytoestrogen‐poor feed and observed a significant increase in granulopoi‐esis, erythropoiesis, and mild macrocytosis at the adult age of 12 wk. Genistein exposure was associated with hypermethylation of certain repetitive elements, which coincided with a significant down‐regulation of estrogen‐responsive genes and genes involved in hematopoi‐esis in bone marrow cells of genistein‐exposed mice, as assessed by microarray technology. Although genistein exposure did not affect global methylation in fetal liver of fetuses at embryonic day 14.5, it accelerated the switch from primitive to definitive erythroid lineage. Taken together, our data demonstrate that prenatal exposure to genistein affects fetal erythropoiesis and exerts lifelong alterations in gene expression and DNA methylation of hematopoietic cells.—Vanhees, K., Coort, S., Ruitjers, E.J. B., Godschalk, R. W. L., van Schooten, F. J., Barjesteh van Waalwijk van Doorn‐Khosrovani, S. Epigenetics: prenatal exposure to genistein leaves a permanent signature on the hematopoietic lineage. FASEB J. 25, 797–807 (2011). www.fasebj.org

Pleiotropic Benefit of Monomeric and Oligomeric Flavanols on Vascular Health - A Randomized Controlled Clinical Pilot Study

Background Cardiovascular diseases are expanding to a major social-economic burden in the Western World and undermine mans deep desire for healthy ageing. Epidemiological studies suggest that flavanol-rich foods (e.g. grapes, wine, chocolate) sustain cardiovascular health. For an evidenced-based application, however, sound clinical data on their efficacy are strongly demanded. Methods In a double-blind, randomized, placebo-controlled intervention study we supplemented 28 male smokers with 200 mg per day of monomeric and oligomeric flavanols (MOF) from grape seeds. At baseline, after 4 and 8 weeks we measured macro- and microvascular function and a cluster of systemic biomarkers for major pathological processes occurring in the vasculature: disturbances in lipid metabolism and cellular redox balance, and activation of inflammatory cells and platelets. Results In the MOF group serum total cholesterol and LDL decreased significantly (P≤0.05) by 5% (n = 11) and 7% (n = 9), respectively in volunteers with elevated baseline levels. Additionally, after 8 weeks the ratio of glutathione to glutathione disulphide in erythrocytes rose from baseline by 22% (n = 15, P<0.05) in MOF supplemented subjects. We also observed that MOF supplementation exerts anti-inflammatory effects in blood towards ex vivo added bacterial endotoxin and significantly reduces expression of inflammatory genes in leukocytes. Conversely, alterations in macro- and microvascular function, platelet aggregation, plasma levels of nitric oxide surrogates, endothelin-1, C-reactive protein, fibrinogen, prostaglandin F2alpha, plasma antioxidant capacity and gene expression levels of antioxidant defense enzymes did not reach statistical significance after 8 weeks MOF supplementation. However, integrating all measured effects into a global, so-called vascular health index revealed a significant improvement of overall vascular health by MOF compared to placebo (P≤0.05). Conclusion Our integrative multi-biomarker approach unveiled the pleiotropic vascular health benefit of an 8 weeks supplementation with 200 mg/d MOF in humans. Trial Registration ClinicalTrials.gov NCT00742287

The flavanol (-)-epicatechin and its metabolites protect against oxidative stress in primary endothelial cells via a direct antioxidant effect

Accumulating evidence suggests that foods rich in flavanols decrease the risk of developing cardiovascular diseases. Attenuation of oxidative stress was suggested to contribute to the cardiovascular benefit of flavanols. Up to now it was unclear whether flavanol metabolites can also protect cells from oxidative stress. The aim of the present study was to determine the potential contribution of several glucuronidated, methylated and sulfated metabolites of (-)-epicatechin (EC) and (+)-catechin (Cat) to the protection of human vascular endothelial cells (HUVECs) against oxidative stress. The relative potency of the tested compounds to scavenge superoxide anion radicals showed that a free catechol moiety in the molecule is important for the direct antioxidant activity. EC and Cat (0.5, 1, 10µM) were potent radical scavengers and provided protection against intracellular oxidative stress induced by hydrogen peroxide. Although the metabolites provided less intracellular protection compared to EC and Cat, the tested methylated and glucuronidated metabolites reduced oxidative stress significantly in HUVECs. Our results indicate that the metabolites have a relevant contribution in the intracellular protection of EC and Cat against oxidative stress. Also, the direct antioxidant activity plays an important role in this protection.

The cocoa flavanol (−)-epicatechin protects the cortisol response

Various health benefits of the cocoa flavanol (-)-epicatechin (EC) have been attributed to its antioxidant and anti-inflammatory potency. In the present study we investigated whether EC is able to prevent deterioration of the anti-inflammatory effect of the glucocorticoid (GC) cortisol in the presence of oxidative stress. It was found that cortisol reduces inflammation in differentiated monocytes. Oxidative stress extinguishes the anti-inflammatory effect of cortisol, leading to cortisol resistance. EC reduces intracellular oxidative stress as well as the development of cortisol resistance. This further deciphers the enigmatic mechanism of EC by which it exerts its anti-inflammatory and antioxidant action. The observed effect of the cocoa flavanol EC will especially be of relevance in pathophysiological conditions with increased oxidative stress and consequential GC resistance and provides a fundament for the rational use of dietary antioxidants.

Food-Derived Bioactives Can Protect the Anti-Inflammatory Activity of Cortisol with Antioxidant-Dependent and -Independent Mechanisms

In chronic inflammatory diseases the anti-inflammatory effect of glucocorticoids (GCs) is often decreased, leading to GC resistance. Inflammation is related with increased levels of reactive oxygen species (ROS), leading to oxidative stress which is thought to contribute to the development of GC resistance. Plant-derived compounds such as flavonoids are known for their ability to protect against ROS. In this exploratory study we screened a broad range of food-derived bioactives for their antioxidant and anti-inflammatory effects in order to investigate whether their antioxidant effects are associated with the ability to preserve the anti-inflammatory effects of cortisol. The anti-inflammatory potency of the tested compounds was assessed by measuring the oxidative stress–induced GC resistance in human macrophage-like cells. Cells were pre-treated with H2O2 (800 µM) with and without bioactives and then exposed to lipopolysaccharides (LPS) (10 ng/mL) and cortisol (100 nM). The level of inflammation was deducted from the concentration of interleukin-8 (IL-8) in the medium. Intracellular oxidative stress was measured using the fluorescent probe 2′,7′-dichlorofluorescein (DCFH). We found that most of the dietary bioactives display antioxidant and anti-inflammatory action through the protection of the cortisol response. All compounds, except for quercetin, revealing antioxidant activity also protect the cortisol response. This indicates that the antioxidant activity of compounds plays an important role in the protection of the GC response. However, next to the antioxidant activity of the bioactives, other mechanisms also seem to be involved in this protective, anti-inflammatory effect.

Etoposide-initiated MLL rearrangements: the pitfalls of inverse PCR

To the Editor: With great interest we read the letter of Marschalek (‘Postprint’; doi: 10.1111/j.1600-0609.2008.01139.x) written in response to the recent publication of Libura et al. (1) in the European Journal of Haematology. In addition to his remarks and suggestions, we would like to share our experience regarding Inverse PCR (IPCR) methodology. In our laboratory, we frequently apply IPCR to detect chromosomal rearrangements in both human and mice. In the past, we have also applied the methodology of Libura et al. (2) to detect MLL translocations in human CD34 cells exposed to bioflavonoids (3). When comparing the nucleotide sequences of chromosomal translocation detected in our samples with those published by Libura et al. (2), we, like Marschalek, noticed that most of what was called MLL duplication was artificially produced by religation of the DNA ends, which were generated by the XbaI restriction enzyme. Similar to some of the duplications which are presented in their recent manuscript (1), the XbaI restriction site was clearly visible at the breakpoint junction in the fusion partner of MLL. In general, most artifacts related to IPCR are generated as a result of intermolecular ligation instead of self-ligation (Fig. 1A). Yet, the artifacts involving restriction digests are the most innocent of all, as they can be easily recognized by nucleotide sequence analysis. The most treacherous artifacts are those which are produced as a result of direct ligation of genomic fragments and resemble true chromosomal rearrangements (Fig. 1B). Our experience has been that pretreatment of DNA with alkaline phosphatase is essential to prevent the manifestation of such artifacts, especially when the cells are exposed to DNA damaging agents or when the DNA is prepared by whole genome amplification. In addition, diluting the DNA (<5 ng ⁄ lL) at the ligation step promotes self-ligation and reduces the number of various ligation-mediated artifacts. Another point of concern is related to the presence of several Alu repeats within the genomic sequence of the MLL gene. These Alu repeats are thought to be responsible for the frequently occurring MLL rearrangements through Alu-Alu recombination. Accordingly, sequencing of the genomic PCR fragments in leukemic patients as well as healthy individuals demonstrates Alu sequences at or near the breakpoints (4–8). Remarkably, direct PCR on unprocessed DNA, using MLL primers which are designed in opposite direction (2), can amplify MLL rearrangements which resemble partial duplication of the gene (S. Barjesteh van Waalwijk van Doorn-Khosrovani, K. Vanhees, E. Ruijters, unpublished data). The same phenomenon is described in the recent manuscript of Libura et al. (1). Whether all the Alu-mediated rearrangements, which have previously been detected using PCR-based methods, without confirmation by southern blot analysis or other non-PCR-based methods, are true genomic events needs further investigation. In principle, MLL rearrangements can be generated in vitro by annealing of different Alu elements to each other because of high nucleotide resemblance (9). Some of the MLL internal rearrangements that have been detected both by Libura et al. (1) and our group (S. Barjesteh van Waalwijk van Doorn-Khosrovani, K. Vanhees, E. Ruijters, unpublished data) are fusion of the two Alu elements in the vicinity of primer MF2 and MR2. These two Alu elements show a very high homology to each other. If the DNA is not digested by the XbaI restriction enzyme (Fig. 1C) a new DNA strand complementary to the upstream Alu element will be made by MR2 primer during the elongation phase. Similarly the downstream Alu element will be extended by MF2. If polymerase terminates within one Alu, the partially amplified DNA can act as a primer and hybridize to the other Alu in the next cycle. Theoretically, the same can occur when MLL is ligated to a different genomic sequence through its upstream XbaI site. The artifact generated in this way will resemble a translocation. The Alu-mediated artifacts can be recognized by their two distinct features: (i) they share a homologous region and therefore the exact fusion site could not be identified and (ii) as annealing of the strands from the two Alu elements is accompanied with multiple mismatches, because of nucleotide differences between them, direct sequencing of the fusion product would demonstrate multiple double peaks at the homologous region, which originate from a mixture of sequences derived from both Alu elements (9). It is therefore necessary to critically analyze the nucleotide sequences of all fusion products, especially those which are believed to be generated by single-strand annealing pathway. Although IPCR seems to have pitfalls, it remains a valuable, powerful and easy method for detecting translocations. The finding that a dose-dependent increase in the number of MLL translocations can be seen by IPCR methodology, after exposure of CD34 cells to doi:10.1111/j.1600-0609.2008.01147.x European Journal of Haematology ISSN 0902-4441