Kimberly Vanhees

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

Intrauterine exposure to flavonoids modifies antioxidant status at adulthood and decreases oxidative stress-induced DNA damage

Maternal intake of flavonoids, known for their antioxidant properties, may affect the offsprings susceptibility to developing chronic diseases at adult age, especially those related to oxidative stress, via developmental programming. Therefore, we supplemented female mice with the flavonoids genistein and quercetin during gestation, to study their effect on the antioxidant capacity of lung and liver of adult offspring. Maternal intake of quercetin increased the expression of Nrf2 and Sod2 in fetal liver at gestational day 14.5. At adult age, in utero exposure to both flavonoids resulted in the increased expression of several enzymatic antioxidant genes, which was more pronounced in the liver than in the adult lung. Moreover, prenatal genistein exposure induced the nonenzymatic antioxidant capacity in the adult lung, partly by increasing glutathione levels. Prenatal exposure to both flavonoids resulted in significantly lower levels of oxidative stress-induced DNA damage in liver only. Our observations lead to the hypothesis that a preemptive trigger of the antioxidant defense system in utero had a persistent effect on antioxidant capacity and as a result decreased oxidative stress-induced DNA damage in the liver.

Prenatal Exposure to Flavonoids: Implication for Cancer Risk

Flavonoids are potent antioxidants, freely available as high-dose dietary supplements. However, they can induce DNA double-strand breaks (DSB) and rearrangements in the mixed-lineage leukemia (MLL) gene, which are frequently observed in childhood leukemia. We hypothesize that a deficient DSB repair, as a result of an Atm mutation, may reinforce the clastogenic effect of dietary flavonoids and increase the frequency of Mll rearrangements. Therefore, we examined the effects of in vitro and transplacental exposure to high, but biological amounts of flavonoids in mice with different genetic capacities for DSB repair (homozygous/heterozygous knock-in for human Atm mutation [Atm-ΔSRI] vs. wild type [wt]). In vitro exposure to genistein/quercetin induced higher numbers of Mll rearrangements in bone marrow cells of Atm-ΔSRI mutant mice compared with wt mice. Subsequently, heterozygous Atm-ΔSRI mice were placed on either a flavonoid-poor or a genistein-enriched (270 mg/kg) or quercetin-enriched (302 mg/kg) feed throughout pregnancy. Prenatal exposure to flavonoids associated with higher frequencies of Mll rearrangements and a slight increase in the incidence of malignancies in DNA repair-deficient mice. These data suggest that prenatal exposure to both genistein and quercetin supplements could increase the risk on Mll rearrangements especially in the presence of compromised DNA repair.

Maternal quercetin intake during pregnancy results in an adapted iron homeostasis at adulthood.

The flavonoid quercetin is a powerful iron chelator, capable of oxidizing heme iron in hemoglobin from Fe(2+) to Fe(3+). Moreover, quercetin crosses the placenta and accumulates in the fetus. Since adaptations made by the fetus to cope with inappropriate nutrition may lead to permanent changes, a relative high intake of quercetin may have detrimental affects later in life. Therefore, we investigated the effects of maternal exposure to quercetin (302 mg/kg feed), starting from 3 days before conception until the end of gestation, on erythropoiesis and iron homeostasis at embryonic day 14.5 and in 12-week old mice. During fetal development, quercetin exposure had no effect on the erythroid lineage switch and concomitant globin switch. However, adult mice prenatally exposed to quercetin had significant increase iron storage in the liver, by upregulating iron-associated cytokine expression (hepcidin, IL-1β, IL-6 and IL-10). These long term changes in gene expression could be mediated through epigenetic modifications, as prenatal quercetin exposure resulted in a modest hypermethylation of repetitive elements. Despite the increased iron levels, oxidative stress was significantly decreased in the liver of these animals as assessed by 8-oxo-dG levels. These data suggest that prenatal quercetin exposure results in increased iron storage, while decreasing oxidative stress induced DNA damage together with a shift towards increased expression of inflammation associated cytokines in the liver at adult age.

Volatile Organic Compounds in Exhaled Air as Novel Marker for Disease Activity in Crohn's Disease: A Metabolomic Approach.

Background:Disappearance of macroscopic mucosal inflammation predicts long-term outcome in Crohn’s disease (CD). It can be assessed by ileocolonoscopy, which is, however, an invasive and expensive procedure. Disease activity indices do not correlate well with endoscopic activity and noninvasive markers have a low sensitivity in subgroups of patients. Volatile organic compounds (VOCs) in breath are of increasing interest as noninvasive markers. The aim of this study was to investigate whether VOCs can accurately differentiate between active CD and remission. Methods:Patients participated in a 1-year follow-up study and Harvey–Bradshaw index, blood, fecal, and breath samples were collected at regular intervals. Patients were stratified into 2 groups: active (fecal calprotectin >250 µg/g) or inactive (Harvey–Bradshaw index <4, C-reactive protein <5 mg/L, and fecal calprotectin <100 µg/g) disease. Breath samples were analyzed by gas chromatography–time-of-flight mass spectrometry. Random forest analyses were used to find the most discriminatory VOCs. Results:Eight hundred thirty-five breath-o-grams were measured, 140 samples were assigned as active, 135 as inactive disease, and 110 samples of healthy controls. A set of 10 discriminatory VOCs correctly predicted active CD in 81.5% and remission in 86.4% (sensitivity 0.81, specificity 0.80, AUC 0.80). These VOCs were combined into a single disease activity score that classified disease activity in more than 60% of the previously undetermined individuals. Conclusions:We showed that VOCs can separate healthy controls and patients with active CD and CD in remission in a real-life cohort. Analysis of exhaled air is an interesting new noninvasive application for monitoring mucosal inflammation in inflammatory bowel disease.

Maternal intake of quercetin during gestation alters ex vivo benzo[a]pyrene metabolism and DNA adduct formation in adult offspring

Variation in xenobiotic metabolism cannot entirely be explained by genetic diversity in metabolic enzymes. We suggest that maternal diet during gestation can contribute to variation in metabolism by creating an in utero environment that shapes the offsprings defence against chemical carcinogens. Therefore, pregnant mice were supplemented with the natural aryl hydrocarbon receptor (AhR) agonist quercetin (1 mmol quercetin/kg feed) until delivery. Next, it was investigated whether the adult offspring at the age of 12 weeks had altered biotransformation of the environmental pollutant benzo[a]pyrene (B[a]P). In utero quercetin exposure resulted in significantly enhanced gene expression of Cyp1a1, Cyp1b1, Nqo1 and Ugt1a6 in liver of foetuses at Day 14.5 of gestation. Despite cessation of supplementation after delivery, altered gene expression persisted into adulthood, but in a tissue- and gender-dependent manner. Expression of Phase I enzymes (Cyp1a1 and Cyp1b1) was up-regulated in the liver of adult female mice in utero exposed to quercetin, whereas expression of Phase II enzymes (Gstp1, Nqo1 and Ugt1a6) was predominantly enhanced in the lung tissue of female mice. Epigenetic mechanisms may contribute to this adapted gene expression, as the repetitive elements (SINEB1) were hypomethylated in liver of female mice prenatally exposed to quercetin. Studies on ex vivo metabolism of B[a]P by lung and liver microsomes showed that the amount of B[a]P-9,10-dehydrodiol, B[a]P-7,8-dihydrodiol and 3-hydroxy-B[a]P did not change, but the amount of unmetabolised B[a]P was significantly lower after incubation with lung microsomes from offspring that received quercetin during gestation. Moreover, ex vivo B[a]P-induced DNA adduct formation was significantly lower for liver microsomes of offspring that were exposed to quercetin during gestation. These results suggest that prenatal diet leads to persistent alterations in Phase I and II enzymes of adult mice and may affect cancer risk.

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

Does Ataxia Telangiectasia Mutated (ATM) protect testicular and germ cell DNA integrity by regulating the redox status

A balanced redox homeostasis in the testis is essential for genetic integrity of sperm. Reactive oxygen species can disturb this balance by oxidation of glutathione, which is regenerated using NADPH, formed by glucose-6-phosphate dehydrogenase (G6PDH). G6PDH is regulated by the Ataxia Telangiectasia Mutated (Atm) protein. Therefore, we studied the redox status and DNA damage in testes and sperm of mice that carried a deletion in Atm. The redox status in heterozygote mice, reflected by glutathione levels and antioxidant capacity, was lower than in wild type mice, and in homozygotes the redox status was even lower. The redox status correlated with oxidative DNA damage that was highest in mice that carried Atm deletions. Surprisingly, G6PDH activity was highest in homozygotes carrying the deletion. These data indicate that defective Atm reduces the redox homeostasis of the testis and genetic integrity of sperm by regulating glutathione levels independently from G6PDH activity.