Maternal exposure to genistein during pregnancy and oxidative DNA damage in testes of male mouse offspring.

Background: Genistein is a dietary supplement with phyto-estrogenic properties. Therefore, high intake of genistein during pregnancy may have adverse effects on the genetic integrity of testes and germ cells of male offspring. In this study, we examined whether maternal exposure to genistein during pregnancy induced oxidative DNA damage in the male germline at adolescence. Methods: Atm-ΔSRI mice have lower glucose-6-phosphate dehydrogenase (G6PDH) activity, which is important for maintaining levels of reduced glutathione and therefore these mice have an increased susceptibility to oxidative stress. Parental heterozygous Atm-ΔSRI mice received a genistein-rich or control diet, after which they were mated to obtain offspring. During pregnancy, mothers remained on the respective diets and after delivery all animals received control diets. Redox status and oxidative DNA damage were assessed in testes and sperm of 12 weeks old male offspring. Gene expression of Cyp1b1, Comt, and Nqo1 was assessed in testes, and DNA methylation as possible mechanism for transmission of effects to later life. Results: Intake of genistein during pregnancy increased oxidative DNA damage in testes of offspring, especially in heterozygous Atm-ΔSRI mice. These increased DNA damage levels coincided with decreased expression of Comt and Nqo1. Heterozygous Atm-ΔSRI mice had higher levels of DNA strand breaks in sperm compared to wild type littermates, and DNA damage was further enhanced by a genistein-rich maternal diet. G6PDH activity was higher in mice with high maternal intake of genistein compared to control diets, suggesting compensation against oxidative stress. A positive correlation was observed between the levels of DNA methylation and oxidative DNA damage in testes. Conclusion: These data indicate that prenatal exposure to genistein altered gene expression and increased DNA damage in testes and sperm of adolescent male offspring. These effects of genistein on DNA damage in later life coincided with alterations in DNA methylation.

The Dynamics of Nucleotide Variants in the Progression from Low-Intermediate Myeloma Precursor Conditions to Multiple Myeloma: Studying Serial Samples with a Targeted Sequencing Approach.

Multiple myeloma (MM), or Kahler's disease, is an incurable plasma cell (PC) cancer in the bone marrow (BM). This malignancy is preceded by one or more asymptomatic precursor conditions, monoclonal gammopathy of undetermined significance (MGUS) and/or smoldering multiple myeloma (SMM). The molecular mechanisms and exact cause of this progression are still not completely understood. In this study, the mutational profile underlying the progression from low-intermediate risk myeloma precursor conditions to MM was studied in serial BM smears. A custom capture-based sequencing platform was developed, including 81 myeloma-related genes. The clonal evolution of single nucleotide variants and short insertions and deletions was studied in serial BM smears from 21 progressed precursor patients with a median time of progression of six years. From the 21 patients, four patients had no variation in one of the 81 studied genes. Interestingly, in 16 of the 17 other patients, at least one variant present in MM was also detected in its precursor BM, even years before progression. Here, the variants were present in the pre-stage at a median of 62 months before progression to MM. Studying these paired BM samples contributes to the knowledge of the evolutionary genetic landscape and provides additional insight into the mutational behavior of mutant clones over time throughout progression.

Whole-genome sequencing reveals progressive versus stable myeloma precursor conditions as two distinct entities.

Multiple myeloma (MM) is consistently preceded by precursor conditions recognized clinically as monoclonal gammopathy of undetermined significance (MGUS) or smoldering myeloma (SMM). We interrogate the whole genome sequence (WGS) profile of 18 MGUS and compare them with those from 14 SMMs and 80 MMs. We show that cases with a non-progressing, clinically stable myeloma precursor condition (n = 15) are characterized by later initiation in the patient's life and by the absence of myeloma defining genomic events including: chromothripsis, templated insertions, mutations in driver genes, aneuploidy, and canonical APOBEC mutational activity. This data provides evidence that WGS can be used to recognize two biologically and clinically distinct myeloma precursor entities that are either progressive or stable.

Raising to the Challenge: Building a Federated Biobank to Accelerate Translational Research-The University Biobank Limburg.

Irreproducibility of research results is one of the major contributing factors to the failure of translating basic research results into tangible bedside progress. To address this, the University Biobank Limburg (UBiLim) was founded by a collaboration between Hasselt University, the Hospital East-Limburg, and the Jessa Hospital. This paper describes the evolution of this process and the barriers encountered on the way. UBiLim evolved from an archival collection over a single-site biobank into a federated structure, supporting translational research at the founding institutions. Currently, UBiLim is a federated biobank, with an established organizational structure and processing, and storage facilities at each of the three sites. All activities are integrated in an ISO15189-accredited Quality Management System and based on (inter)national biobank guidelines. Common methods for processing and storage of a plethora of sample types, suitable for state-of-the-art applications, were validated and implemented. Because the biobank is embedded in two hospitals, the request of researchers to include certain sample types or enroll specific patient groups can quickly be met. Funding has been a major challenge in each step of its evolution and remains the biggest issue for long-term biobank sustainability. To a lesser extent, the Belgian legislation and the operational cost of information management system are also concerns for smooth biobank operations. Nonetheless, UBiLim serves as a facilitator and accelerator for translational research in the Limburg area of Belgium that, given the fields of research, may have an impact on international patient care.

Archival May-Grünwald-Giemsa-Stained Bone Marrow Smears Are an Eligible Source for Molecular DNA Research.

Biobanking is increasingly important in studying complex heterogeneous diseases. Therefore, it is essential to ensure the sample quality after long-term storage for reliable downstream analyses. The Clinical Biobank of the Jessa Hospital and the University Biobank Limburg (UBiLim) hold a continuously growing collection of hematological samples, including May-Grünwald-Giemsa (MGG)- and Perls' Prussian Blue (PPB)-stained bone marrow (BM) smears, stored at room temperature (RT) for up to 20 years. In this study, we investigated the effect of short- and long-term storage on the quality of DNA and RNA extracted from these BM smears to assess their fitness-for-purpose in downstream molecular applications, including agarose gel electrophoresis, bio-analyzer analysis, quantitative polymerase chain reaction (qPCR), and targeted next-generation sequencing (NGS). The RNA quality was very low for all samples, independent of storage time or staining method. The DNA from PPB-stained BM smears was already degraded after 1 year of storage and correspondingly could not be used for reliable downstream molecular analysis. In contrast, DNA extracted from MGG-stained BM smears stored for up to 10 years was able to generate high-quality data in qPCR and targeted NGS analyses. Longer storage periods (>15 years) of these samples revealed a high degree of degradation and a significant amount of DNA transitions and transversions. In conclusion, the DNA extracted from archival MGG-stained BM smears with a storage time up to at least 10 years was qualitatively good and fit for downstream analysis, including targeted NGS. This indicates that these samples are an eligible source for molecular DNA research and for studying complex diseases.

Prognostic Biomarkers in the Progression From MGUS to Multiple Myeloma: A Systematic Review.

Multiple myeloma (MM), characterized by malignant plasma cells in the bone marrow, is consistently preceded by asymptomatic premalignant stage monoclonal gammopathy of undetermined significance (MGUS). These MGUS patients have an annual risk of 1% to progress to MM. Clinical, imaging, and genomic (genetic and epigenetic) factors were identified, whose presence increased the risk of progression from MGUS to MM. In this systematic review we summarize the currently identified clinical, imaging, and genomic biomarkers suggested to increase the progression risk or shown to be differentially expressed/present between both cohorts of patients. Despite the wide range of proposed markers, there are still no reliable biomarkers to individually predict which MGUS patient will progress to MM and which will not. Research on biomarkers in the progression from MGUS to MM will give more insight in the unknown pathogenesis of this hematological malignancy. This would improve research by elucidating new pathways and potential therapeutic targets as well as clinical management by closer follow-up and earlier treatment of high-risk MGUS patients.

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.

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.

You are what you eat, and so are your children: the impact of micronutrients on the epigenetic programming of offspring.

The research field of fetal programming has developed tremendously over the years and increasing knowledge suggests that both maternal and paternal unbalanced diet can have long-lasting effects on the health of offspring. Studies implicate that macronutrients play an important role in fetal programming, although the importance of micronutrients is also becoming increasingly apparent. Folic acid and vitamins B2, B6 and B12 are essential for one-carbon metabolism and are involved in DNA methylation. They can therefore influence the programming of the offspring's epigenome. Also, other micronutrients such as vitamins A and C, iron, chromium, zinc and flavonoids play a role in fetal programming. Since it is estimated that approximately 78 % of pregnant women in the US take vitamin supplements during pregnancy, more attention should be given to the long-term effects of these supplements on offspring. In this review we address several different studies which illustrate that an unbalanced diet prior and during pregnancy, regarding the intake of micronutrients of both mother and father, can have long-lasting effects on the health of adult offspring.

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 offspring's 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.

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-1ß, 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.

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.

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 granulopoiesis, 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 hematopoiesis 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.