Galacto-oligosaccharides supplementation in prefrail older and healthy adults increased faecal bifidobacteria, but did not impact immune function and oxidative stress.

BACKGROUND & AIMS: Ageing is associated with an increased risk of frailty, intestinal microbiota perturbations, immunosenescence and oxidative stress. Prebiotics such as galacto-oligosaccharides (GOS) may ameliorate these ageing-related alterations. We aimed to compare the faecal microbiota composition, metabolite production, immune and oxidative stress markers in prefrail elderly and younger adults, and investigate the effects of GOS supplementation in both groups. METHODS: In a randomised controlled cross-over study, 20 prefrail elderly and 24 healthy adults received 21.6 g/day Biotis™ GOS (containing 15.0 g/day GOS) or placebo. Faecal 16S rRNA gene-based microbiota and short-chain fatty acids were analysed at 0, 1 and 4 weeks of intervention.Volatile organic compounds were analysed in breath, and stimulated cytokine production, CRP, malondialdehyde, trolox equivalent antioxidant capacity (TEAC) and uric acid (UA) in blood at 0 and 4 weeks. RESULTS: Principle coordinate analysis showed differences in microbial composition between elderly and adults (P≤0.05), with elderly having lower bifidobacteria (P≤0.033) at baseline. In both groups, GOS affected microbiota composition (P≤0.05), accompanied by increases in bifidobacteria (P<0.001) and decreased microbial diversity (P≤0.023). Faecal and breath metabolites, immune and oxidative stress markers neither differed between groups (P ≥ 0.125) nor were affected by GOS (P ≥ 0.236). TEAC values corrected for UA were higher in elderly versus adults (P<0.001), but not different between interventions (P ≥ 0.455). CONCLUSIONS: Elderly showed lower faecal bifidobacterial (relative) abundance than adults, which increased after GOS intake in both groups. Faecal and breath metabolites, parameters of immune function and oxidative stress were not different at baseline, and not impacted by GOS supplementation. CLINICALTRIALS. GOV WITH STUDY ID NUMBER: NCT03077529.

Sugar Beet Pectin Supplementation Did Not Alter Profiles of Fecal Microbiota and Exhaled Breath in Healthy Young Adults and Healthy Elderly.

Aging is accompanied with increased frailty and comorbidities, which is potentially associated with microbiome perturbations. Dietary fibers could contribute to healthy aging by beneficially impacting gut microbiota and metabolite profiles. We aimed to compare young adults with elderly and investigate the effect of pectin supplementation on fecal microbiota composition, short chain fatty acids (SCFAs), and exhaled volatile organic compounds (VOCs) while using a randomized, double-blind, placebo-controlled parallel design. Fifty-two young adults and 48 elderly consumed 15 g/day sugar beet pectin or maltodextrin for four weeks. Fecal and exhaled breath samples were collected before and after the intervention period. Fecal samples were used for microbiota profiling by 16S rRNA gene amplicon sequencing, and for analysis of SCFAs by gas chromatography (GC). Breath was used for VOC analysis by GC-tof-MS. Young adults and elderly showed similar fecal SCFA and exhaled VOC profiles. Additionally, fecal microbiota profiles were similar, with five genera significantly different in relative abundance. Pectin supplementation did not significantly alter fecal microbiota, SCFA or exhaled VOC profiles in elderly or young adults. In conclusion, aside from some minor differences in microbial composition, healthy elderly and young adults showed comparable fecal microbiota composition and activity, which were not altered by pectin supplementation.

The shifting perception on antioxidants: the case of vitamin E and ß-carotene.

Antioxidants are vital for aerobic life, and for decades the expectations of antioxidants as health promoting agents were very high. However, relatively recent meta-analyses of clinical studies show that supplementation of antioxidants does not result in the presumed health benefit, but is associated with increased mortality. The dilemma that still needs to be solved is: what are antioxidants in the end, healthy or toxic? We have evaluated this dilemma by examining the presumed health effects of two individual antioxidants with opposite images i.e. the "poisonous" ß-carotene and the "wholesome" vitamin E and focused on one aspect, namely their role in inducing BPDE-DNA adducts. It appears that both antioxidants promote DNA adduct formation indirectly by inhibition of the protective enzyme glutathione-S-transferase π (GST π). Despite their opposite image, both antioxidants display a similar type of toxicity. It is concluded that, in the appreciation of antioxidants, first their benefits should be identified and substantiated by elucidating their molecular mechanism. Subsequently, the risks should be identified including the molecular mechanism. The optimal benefit-risk ratio has to be determined for each antioxidant and each individual separately, also considering the dose.

Selenoprotein gene variants, toenail selenium levels, and risk for advanced prostate cancer.

Lower selenium levels have been associated with increased risk of prostate cancer (PCa), and genetic variation in the selenoprotein genes selenoprotein P (SEPP1) and glutathione peroxidase 1 (GPX1) is thought to modify this relationship. We investigated whether the association between toenail selenium levels and advanced PCa risk in the prospective Netherlands Cohort Study is modified by common genetic variation in SEPP1 and GPX1. Toenail clippings were used to determine selenium levels and to isolate DNA for genotyping. This case-cohort study, which included 817 case subjects with advanced PCa and 1048 subcohort members, was analyzed with Cox regression models. All statistical tests were two-sided. Three genetic variants were associated with advanced (stage III/IV or IV) PCa risk: SEPP1 rs7579 (lower risk; P trend = .01), GPX1 rs17650792 (higher risk; P trend = .03), and GPX1 rs1800668 (lower risk; P trend = .005). Toenail selenium levels were inversely associated with advanced PCa risk, independently of common genetic variation in SEPP1 and GPX1.

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.

Advanced prostate cancer risk in relation to toenail selenium levels.

BACKGROUND: Selenium may prevent advanced prostate cancer (PCa), but most studies on this topic were conducted in populations with moderate to high selenium status. We investigated the association of toenail selenium, reflecting long-term selenium exposure, and advanced PCa risk in a population from the Netherlands where low selenium status is widespread. METHODS: The analysis was conducted in the prospective Netherlands Cohort Study, which included 58 279 men aged 55 to 69 years at baseline in 1986. All cohort members completed a baseline questionnaire, and approximately 79% of participants provided toenail clippings, which were used for toenail selenium measurements using instrumental neutron activation analysis. Incident advanced PCa case subjects from the entire cohort were identified during 17.3 years of follow-up. The study employed a case-cohort design for which a random subcohort was sampled at baseline. Hazard ratios and 95% confidence intervals (CIs) were estimated using Cox proportional hazards regression models. All tests were two-sided. RESULTS: Complete toenail selenium data were available for 898 advanced (International Union Against Cancer stage III/IV) PCa case subjects and 1176 subcohort members. The average toenail selenium concentration of subcohort members was 0.550 µg/g. Toenail selenium was associated with a reduced risk of advanced PCa; adjusted hazard ratio for the highest vs lowest quintile was 0.37 (95% CI = 0.27 to 0.51; P trend < .001). For stage IV PCa, men in the highest vs lowest quintile of toenail selenium had an adjusted hazard ratio of 0.30 (95% CI = 0.21 to 0.45; P trend < .001). CONCLUSIONS: Toenail selenium was associated with a substantial decrease in risk of advanced PCa.

Dietary flavonoid intake, black tea consumption, and risk of overall and advanced stage prostate cancer.

Flavonoids are natural antioxidants found in various foods, and a major source is black tea. Some experimental evidence indicates that flavonoids could prevent prostate cancer. We investigated the associations between flavonoid intake, black tea consumption, and prostate cancer risk in the Netherlands Cohort study, which includes 58,279 men who provided detailed baseline information on several cancer risk factors. From 1986 to 2003, 3,362 prostate cancers were identified, including 1,164 advanced (stage III/IV) cancers. Cox proportional hazards regression using the case-cohort approach was used to estimate hazard ratios and 95% confidence intervals. Intake of total catechin, epicatechin, kaempferol, and myricetin and consumption of black tea were associated with a decreased risk of stage III/IV or stage IV prostate cancer. Hazard ratios of stage III/IV and stage IV prostate cancer for the highest versus the lowest category of black tea consumption (≥5 versus ≤1 cups/day) were 0.75 (95% confidence interval: 0.59, 0.97) and 0.67 (95% confidence interval: 0.50, 0.91), respectively. No associations were observed for overall and nonadvanced prostate cancer. In conclusion, dietary flavonoid intake and black tea consumption were associated with a decreased risk of advanced stage prostate cancer.

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.

Organ specificity of beta-carotene induced lung gene-expression changes in Bcmo1-/- mice.

SCOPE: Whole genome transcriptome analysis of male and female beta-carotene 15,15'-monooxygenase knockout (Bcmo1(-/-) ) and Bcmo1(+/+) (wild-type) mice with or without 14 wk of BC supplementation was done. We previously showed that only 1.8% of the genes regulated by BC in lung were also regulated in liver and inguinal white adipose tissue (iWAT), suggesting lung specific responses. Here, we explicitly questioned the lung specificity. METHODS AND RESULTS: We show that BC supplementation resulted in an opposite direction of gene-regulation in male compared to female Bcmo1(-/-) mice in lung, liver, and iWAT. This supports a systemic effect of BC on steroid hormone metabolism mediated responses. Lung, liver, and iWAT of female Bcmo1(-/-) mice showed an increased inflammatory response, which was counteracted by supplementation of BC. This supports a genotype dependent increased sensitivity of female mice for vitamin A deficiency. Finally, the effect of BC on Wnt signaling in male Bcmo1(-/-) mice was examined. Frizzled homolog 6 (Fzd6) downregulation was seen in all three tissues. Collagen triple helix containing 1 (Cthrc1) downregulation was seen in lung tissue only, suggesting specificity. Upregulation of genes involved in oxygen sensing was seen in lung and iWAT, while protocadherin upregulation was only seen in lung. CONCLUSION: Our results demonstrate that effects of BC are strongly sex dependent. While effects of BC on hormone metabolism mediated responses and inflammation are systemic, effects on Wnt signaling may be lung specific.

Gene expression response of mouse lung, liver and white adipose tissue to ß-carotene supplementation, knockout of Bcmo1 and sex.

SCOPE: Little information is available on differences, commonalities and especially interactions in overall gene expression responses as a result of diet, differences in sex (male and female) and effects induced by differences in metabolism. Moreover, it is unknown whether such effects are tissue specific. METHODS AND RESULTS: We investigated the gene expression effects induced by ß-carotene (BC) supplementation, knockout of ß-carotene 15,15'-monooxygenase 1 (Bcmo1) and differences between male and female mice in lung, liver and inguinal white adipose tissue (iWAT). Unsupervised principal component analysis showed that lung gene expression was most affected by knockout of Bcmo1. Liver was most affected by knockout of Bcmo1 and differences in sex. iWAT was most affected by differences in sex. Hardly any genes were commonly influenced by BC among the three tissues. The effect of BC supplementation and knockout of Bcmo1 were relatively sex specific, especially in iWAT. CONCLUSION: These data demonstrate that gene expression differences induced by BC are limited to the tissue and sex that is analyzed, and that differences in metabolism induced by for example single nucleotide polymorphisms, should be taken into account as much as possible. Moreover, our results indicate that translation from one tissue to the other should be done with caution for any nutritional intervention.

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.

Beta-carotene affects gene expression in lungs of male and female Bcmo1 (-/-) mice in opposite directions.

Molecular mechanisms triggered by high dietary beta-carotene (BC) intake in lung are largely unknown. We performed microarray gene expression analysis on lung tissue of BC supplemented beta-carotene 15,15'-monooxygenase 1 knockout (Bcmo1 (-/-)) mice, which are-like humans-able to accumulate BC. Our main observation was that the genes were regulated in an opposite direction in male and female Bcmo1 (-/-) mice by BC. The steroid biosynthetic pathway was overrepresented in BC-supplemented male Bcmo1 (-/-) mice. Testosterone levels were higher after BC supplementation only in Bcmo1 (-/-) mice, which had, unlike wild-type (Bcmo1 (+/+)) mice, large variations. We hypothesize that BC possibly affects hormone synthesis or metabolism. Since sex hormones influence lung cancer risk, these data might contribute to an explanation for the previously found increased lung cancer risk after BC supplementation (ATBC and CARET studies). Moreover, effects of BC may depend on the presence of frequent human BCMO1 polymorphisms, since these effects were not found in wild-type mice.

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.

Downregulation of Fzd6 and Cthrc1 and upregulation of olfactory receptors and protocadherins by dietary beta-carotene in lungs of Bcmo1-/- mice.

An ongoing controversy exists on beneficial versus harmful effects of high beta-carotene (BC) intake, especially for the lung. To elucidate potential mechanisms, we studied effects of BC on lung gene expression. We used a beta-carotene 15,15'-monooxygenase 1 (Bcmo1) knockout mouse (Bcmo1(-/-)) model, unable to convert BC to retinoids, and wild-type mice (Bcmo1(+/+)) mice to dissect the effects of intact BC from effects of BC metabolites. As expected, BC supplementation resulted in a higher BC accumulation in lungs of Bcmo1(-/-) mice than in lungs of Bcmo1(+/+) mice. Whole mouse genome transcriptome analysis on lung tissue revealed that more genes were regulated in Bcmo1(-/-) mice than Bcmo1(+/+) mice upon BC supplementation. Frizzled homolog 6 (Fzd6) and collagen triple helix repeat containing 1 (Cthrc1) were significantly downregulated (fold changes -2.99 and -2.60, respectively, false discovery rate < 0.05) by BC in Bcmo1(-/-). Moreover, many olfactory receptors and many members of the protocadherin family were upregulated. Since both olfactory receptors and protocadherins have an important function in sensory nerves and Fzd6 and Cthrc1 are important in stem cell development, we hypothesize that BC might have an effect on the highly innervated pulmonary neuroendocrine cell (PNEC) cluster. PNECs are highly associated with sensory nerves and are important cells in the control of stem cells. A role for BC in the innervated PNEC cluster might be of particular importance in smoke-induced carcinogenesis since PNEC-derived lung cancer is highly associated with tobacco smoke.

Knockout of the Bcmo1 gene results in an inflammatory response in female lung, which is suppressed by dietary beta-carotene.

Beta-carotene 15,15'-monooxygenase 1 knockout (Bcmo1 (-/-)) mice accumulate beta-carotene (BC) similarly to humans, whereas wild-type (Bcmo1 (+/+)) mice efficiently cleave BC. Bcmo1 (-/-) mice are therefore suitable to investigate BC-induced alterations in gene expression in lung, assessed by microarray analysis. Bcmo1 (-/-) mice receiving control diet had increased expression of inflammatory genes as compared to BC-supplemented Bcmo1 (-/-) mice and Bcmo1 (+/+) mice that received either control or BC-supplemented diets. Differential gene expression in Bcmo1 (-/-) mice was confirmed by real-time quantitative PCR. Histochemical analysis indeed showed an increase in inflammatory cells in lungs of control Bcmo1 (-/-) mice. Supported by metabolite and gene-expression data, we hypothesize that the increased inflammatory response is due to an altered BC metabolism, resulting in an increased vitamin A requirement in Bcmo1 (-/-) mice. This suggests that effects of BC may depend on inter-individual variations in BC-metabolizing enzymes, such as the frequently occurring human polymorphisms in BCMO1.

The effect of oxidative stress on nucleotide-excision repair in colon tissue of newborn piglets.

Nucleotide-excision repair (NER) is important for the maintenance of genomic integrity and to prevent the onset of carcinogenesis. Oxidative stress was previously found to inhibit NER in vitro, and dietary antioxidants could thus protect DNA not only by reducing levels of oxidative DNA damage, but also by protecting NER against oxidative stress-induced inhibition. To obtain further insight in the relation between oxidative stress and NER activity in vivo, oxidative stress was induced in newborn piglets by means of intra-muscular injection of iron (200mg) at day 3 after birth. Indeed, injection of iron significantly increased several markers of oxidative stress, such as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) levels in colon DNA and urinary excretion of 8-oxo-7,8-dihydroguanine (8-oxoGua). In parallel, the influence of maternal supplementation with an antioxidant-enriched diet was investigated in their offspring. Supplementation resulted in reduced iron concentrations in the colon (P=0.004) at day 7 and a 40% reduction of 8-oxodG in colon DNA (P=0.044) at day 14 after birth. NER capacity in animals that did not receive antioxidants was significantly reduced to 32% at day 7 compared with the initial NER capacity on day 1 after birth. This reduction in NER capacity was less pronounced in antioxidant-supplemented piglets (69%). Overall, these data indicate that NER can be reduced by oxidative stress in vivo, which can be compensated for by antioxidant supplementation.

Modulation of nucleotide excision repair in human lymphocytes by genetic and dietary factors.

Gene-environment interactions determine inter-individual variations in nucleotide excision repair (NER) capacity. Oxidative stress was previously found to inhibit NER, thus supplementation with dietary antioxidants could prevent this inhibition, especially in genetically susceptible subjects. To study the effects of genetic polymorphisms in NER-related genes and dietary intake of antioxidants on an individual's NER capacity, lymphocytes of 168 subjects were isolated before and after a 4-week blueberry and apple juice intervention. Twelve genetic polymorphisms in NER genes XPA, XPC, ERCC1, ERCC2, ERCC5, ERCC6 and RAD23B were assessed by multiplex PCR with single base extension. Based on specific genotype combinations, a subset of individuals (n 36) was selected for phenotypical assessment of NER capacity, which was significantly affected by the total sum of low-activity alleles (P = 0.027). The single polymorphism XPA G23A was the strongest predictor of NER capacity (P = 0.002); carriers of low-activity alleles AA had about three times lower NER capacity than XPA GG carriers. NER capacity assessed before and after intervention correlated significantly (R(2) 0.69; P < 0.001), indicating that inter-individual differences in NER capacity are maintained over 4 weeks. Although the intervention increased plasma trolox equivalent antioxidant capacity from 791 (SE 6.61) to 805 (SE 7.90) microm (P = 0.032), on average it did not affect NER capacity. Nonetheless, carriers of twelve or more low-activity alleles seemed to benefit from the intervention (P = 0.013). Among these, carriers of the variant allele for RAD23B Ala249Val showed improved NER capacity upon intervention (P = 0.020). In conclusion, improved NER capacity upon dietary intervention was detected in individuals carrying multiple low-activity alleles. The XPA G23A polymorphism might be a predictor for NER capacity.

Beta-carotene affects oxidative stress-related DNA damage in lung epithelial cells and in ferret lung.

Beta-carotene (BC) was found to enhance lung cancer risk in smokers. This adverse effect was unexpected because BC was thought to act as an anti-oxidant against cigarette smoke-derived radicals. These radicals can directly or indirectly damage DNA, leading to the formation of pro-mutagenic DNA lesions such as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and 3-(2-deoxy-beta-D-erythro-pentafuranosyl)pyrimido[1,2-alpha]purin-10(3H)-one deoxyguanosine (M(1)dG). Later, it was suggested that high concentrations of BC could also result in pro-oxidant effects. Therefore, we investigated whether high but physiologically feasible concentrations of BC were able to alter (i) the formation of radicals in vitro assessed by electron spin resonance spectroscopy, (ii) the levels of 8-oxo-dG and M(1)dG in vitro in lung epithelial cells after incubation with hydrogen peroxide (H(2)O(2)) and the smoke-derived carcinogen benzo[a]pyrene (B[a]P) and (iii) the levels of 8-oxo-dG and M(1)dG in vivo in ferrets' lung after chronic exposure to B[a]P. BC increased in vitro hydroxyl radical formation in the Fenton reaction but inhibited the formation of carbon-centered radicals. Similarly, BC was able to enhance 8-oxo-dG in vitro in lung epithelial cells. On the other hand, BC significantly inhibited M(1)dG formation in lung epithelial cells, especially after induction of M(1)dG by H(2)O(2) or B[a]P. Finally, BC supplementation of ferrets also resulted in a significant decrease in M(1)dG, but in contrast to the in vitro experiments, no effect was observed on 8-oxo-dG levels, probably because of increased base excision repair capacities as assessed by a modified comet assay. These data indicate that the fate of BC being a pro- or anti-oxidant strongly depends on the type of radical involved.

Impact of multiple genetic polymorphisms on effects of a 4-week blueberry juice intervention on ex vivo induced lymphocytic DNA damage in human volunteers.

Consumption of fruits and vegetables has been associated with a decrease in cancer incidence and cardiovascular disease, presumably caused by antioxidants. We designed a human intervention study to assess antioxidative and possible anti-genotoxic properties of fruit-borne antioxidants. We hypothesized that individuals bearing genetic polymorphisms for genes related to quercetin metabolism, benzo[a]pyrene metabolism, oxidative stress and DNA repair differ in their response to DNA protective effects of increased antioxidant intake. In the present study, 168 healthy volunteers consumed a blueberry/apple juice that provided 97 mg quercetin and 16 mg ascorbic acid a day. After a 4-week intervention period, plasma concentrations of quercetin and ascorbic acid and trolox equivalent antioxidant capacity (TEAC) were significantly increased. Further, we found 20% protection (P < 0.01) against ex vivo H(2)O(2)-provoked oxidative DNA damage, measured by comet assay. However, the level of ex vivo induced benzo[a]pyrene-diol-epoxide (BPDE)-DNA adducts was 28% increased upon intervention (P < 0.01). Statistical analysis of 34 biologically relevant genetic polymorphisms revealed that six significantly influenced the outcome of the intervention. Lymphocytes from individuals bearing variant genotype for Cyp1B1 5 seemed to benefit more than wild-types from DNA damage-protecting effects upon intervention. Variants for COMT tended to benefit less or even experienced detrimental effects from intervention. With respect to GSTT1, the effect is ambiguous; variants respond better in terms of intervention-related increase in TEAC, but wild-types benefit more from its protecting effects against oxidative DNA damage. We conclude that genotyping for relevant polymorphisms enables selecting subgroups among the general population that benefit more of DNA damage-modulating effects of micronutrients.

Dietary flavonoids induce MLL translocations in primary human CD34+ cells.

Genetic abnormalities leading to infant leukemias already occur during fetal development and often involve rearrangements of the mixed-lineage leukemia (MLL) gene. These rearrangements resemble the aberrations observed in therapy-related leukemias following treatment with topoisomerase II (topoII)-inhibiting agents such as etoposide. Since flavonoids are potent topoII inhibitors, we examined the role of three widely consumed dietary flavonoids (quercetin, genistein and kaempferol) on the development of MLL rearrangements in primary human CD34(+) cells. Using the neutral Comet assay, we demonstrated a dose-dependent double-strand break (DSB) formation after exposure to flavonoids. An incorrect repair of these DSBs resulted in chromosomal translocations that co-localized with those identified in infant leukemias. Most of these translocations were formed by microhomology-mediated end joining. Moreover, in all but one translocation, SINE/Alu or LINE/L1 repetitive elements were present in at least one side of the breakpoint junction. Beside MLL translocations, fluorescence in situ hybridization analysis demonstrated monosomy or trisomy of MLL in 8-10% of the quercetin-exposed CD34(+) cells. Our study demonstrates that biologically relevant concentrations of flavonoids can induce MLL abnormalities in primary hematopoietic progenitor cells. This is particularly alarming knowing that the differences in metabolism and excretion rate between mother and fetus can lead to a higher flavonoid concentration on the fetal side. Therefore, it is important to raise public awareness and set guidelines for marketing flavonoid supplements to reduce the risk of infant leukemias.