There is ample scientific evidence suggesting that the health benefits of eating the right amounts of a variety of vegetables and fruit are the consequence of the combined action of different ...phytochemicals. The present review provides an update of the scientific literature on additive and synergistic effects of mixtures of phytochemicals. Most research has been carried out in in vitro systems in which synergistic or additive effects have been established on the level of cell proliferation, apoptosis, antioxidant capacity, and tumor incidence, accompanied by changes in gene and protein expression in relevant pathways underlying molecular mechanisms of disease prevention. The number of human dietary intervention studies investigating complex mixtures of phytochemicals is relatively small, but showing promising results. These studies have demonstrated that combining transcriptomic data with phenotypic markers provide insight into the relevant cellular processes which contribute to the antioxidant response of complex mixtures of phytochemicals. Future studies should be designed as short‐term studies testing different combinations of vegetables and fruit, in which markers for disease outcome as well as molecular (‘omics)‐markers and genetic variability between subjects are included. This will create new opportunities for food innovation and the development of more personalized strategies for prevention of chronic diseases.
Complex mixtures of bioactive compounds (phytochemicals) in vegetables and fruits are thought to be responsible for their beneficial health effects. Future studies should test different combinations of vegetables and fruit, in which markers for disease outcome, molecular (‘omics)‐markers and genetic variability included. This will create opportunities for food innovation and the development of personalized strategies for prevention of chronic diseases.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Abstract Chronic exposure to aflatoxin B1 (AFB1) has, in certain regions in the world, been strongly associated with hepatocellular carcinoma (HCC) development. AFB1 is a very potent hepatotoxic and ...carcinogenic mycotoxin which is frequently reported as a food contaminant. Epigenetic modifications provoked by environmental exposures, such as AFB1, may create a persistent epigenetic footprint. Deregulation of epigenetic mechanisms has actually been reported in HCC patients following AFB1 exposure; however, no attempts have yet been made to investigate early effects on the epigenome level which may be persistent on longer term, thereby possibly initiating carcinogenic events. In this study, we aim to identify methyl DNA-mRNA-interactions representative for a persistent epigenetic footprint associated with the early onset of AFB1-induced HCC. For this, primary human hepatocytes were exposed to 0.3 μM of AFB1 for 5 days. Persistent epigenetic effects were measured 3 days after terminating the carcinogenic exposure. Whole genome DNA methylation changes and whole genome transcriptomic analysis were analyzed applying microarray technologies, and cross-omics interactions were evaluated. Upon combining transcriptomics data with results on DNA methylation, a range of persistent hyper- and hypo-methylated genes was identified which also appeared affected on the transcriptome level. For six of the hypo-methylated and up-regulated genes, namely TXNRD1, PCNA, CCNK, DIAPH3, RAB27A and HIST1H2BF, a clear role in carcinogenic events could be identified. This study is the first to report on a carcinogen-induced persistent impact on the epigenetic footprint in relation with the transcriptome which could be indicative for the early onset of AFB1-related development of HCC.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Diet is an important determinant of overall health, and has been linked to the risk of various cancers. To understand the mechanisms involved, transcriptomic responses from human intervention studies ...are very informative. However, gene expression analysis of human biopsy material only represents the average profile of a mixture of cell types that can mask more subtle, but relevant cell-specific changes. Here, we use the CIBERSORTx algorithm to generate single-cell gene expression from human multicellular colon tissue. We applied the CIBERSORTx to microarray data from the PHYTOME study, which investigated the effects of different types of meat on transcriptional and biomarker changes relevant to colorectal cancer (CRC) risk. First, we used single-cell mRNA sequencing data from healthy colon tissue to generate a novel signature matrix in CIBERSORTx, then we determined the proportions and gene expression of each separate cell type. After comparison, cell proportion analysis showed a continuous upward trend in the abundance of goblet cells and stem cells, and a continuous downward trend in transit amplifying cells after the addition of phytochemicals in red meat products. The dietary intervention influenced the expression of genes involved in the growth and division of stem cells, the metabolism and detoxification of enterocytes, the translation and glycosylation of goblet cells, and the inflammatory response of innate lymphoid cells. These results show that our approach offers novel insights into the heterogeneous gene expression responses of different cell types in colon tissue during a dietary intervention.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Arsenic is an established human carcinogen, but the mechanisms through which it contributes to for instance lung cancer development are still unclear. As arsenic is methylated during its metabolism, ...it may interfere with the DNA methylation process, and is therefore considered to be an epigenetic carcinogen. In the present study, we hypothesize that arsenic is able to induce DNA methylation changes, which lead to changes in specific gene expression, in pathways associated with lung cancer promotion and progression. A549 human adenocarcinoma lung cells were exposed to a low (0.08 µM), intermediate (0.4 µM) and high (2 µM) concentration of sodium arsenite for 1, 2 and 8 weeks. DNA was isolated for whole-genome DNA methylation analyses using NimbleGen 2.1 M deluxe promoter arrays. In addition, RNA was isolated for whole-genome transcriptomic analysis using Affymetrix microarrays. Arsenic modulated DNA methylation and expression levels of hundreds of genes in a dose-dependent and time-dependent manner. By combining whole-genome DNA methylation and gene expression data with possibly involved transcription factors, a large molecular interaction network was created based on transcription factor-target gene pairs, consisting of 216 genes. A tumor protein p53 (
TP53
) subnetwork was identified, showing the interactions of
TP53
with other genes affected by arsenic. Furthermore, multiple other new genes were discovered showing altered DNA methylation and gene expression. In particular, arsenic modulated genes which function as transcription factor, thereby affecting target genes which are known to play a role in lung cancer promotion and progression.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Consumption of nitrate-rich beetroot juice (BRJ) by athletes induces a number of beneficial physiological health effects, which are linked to the formation of nitric oxide (NO) from nitrate. However, ...following a secondary pathway, NO may also lead to the formation of
-nitroso compounds (NOCs), which are known to be carcinogenic in 39 animal species. The extent of the formation of NOCs is modulated by various other dietary factors, such as vitamin C. The present study investigates the endogenous formation of NOCs after BRJ intake and the impact of vitamin C on urinary NOC excretion. In a randomized, controlled trial, 29 healthy recreationally active volunteers ingested BRJ with or without additional vitamin C supplements for one week. A significant increase of urinary apparent total
-nitroso Compounds (ATNC) was found after one dose (5 to 47 nmol/mmol:
< 0.0001) and a further increase was found after seven consecutive doses of BRJ (104 nmol/mmol:
< 0.0001). Vitamin C supplementation inhibited ATNC increase after one dose (16 compared to 72 nmol/mmol,
< 0.01), but not after seven daily doses. This is the first study that shows that BRJ supplementation leads to an increase in formation of potentially carcinogenic NOCs. In order to protect athlete's health, it is therefore important to be cautious with chronic use of BRJ to enhance sports performances.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Blueberries contain many different phytochemicals which might be responsible for their disease preventive properties. In a previously conducted human dietary intervention study, we showed that a ...4-week intervention with blueberry⁻apple juice protected the participants against oxidative stress and modulated expression of genes involved in different genetic pathways contributing to the antioxidant response. The present study investigates the effect of different blueberry varieties (Elliot, Draper, Bluecrop, and Aurora, and the blueberry⁻apple juice from our previous human dietary intervention study), and four different single compounds (vitamin C, peonidin, cyanidin, and quercetin) on antioxidant capacity and gene expression changes in colonic cells in vitro, and compares the outcome with the earlier in vivo findings. The results demonstrate that all blueberry varieties as well as the blueberry⁻apple juice were more effective in reducing oxidative stress as compared to the single compounds (e.g., DNA strand break reduction: EC
: Elliot 8.3 mg/mL, Aurora and Draper 11.9 mg/mL, blueberry⁻apple juice 12.3 mg/mL, and Bluecrop 12.7 mg/mL; single compounds). In addition, the gene expression profiles (consisting of 18 selected genes from the in vivo study) induced by the blueberry varieties were more similar to the profile of the human intervention study (range 44⁻78%). The blueberry variety Elliot showed the strongest and most similar effects, almost 80% of gene expression modulations were similar compared to the in vivo results. From the single compounds (range 17⁻44%), quercetin induced the most comparable gene expression changes, i.e., 44%. This approach could be useful in agriculture for identifying crop varieties containing combinations of phytochemicals which show optimal preventive capacities.
Valproic acid (VPA) is one of the most widely prescribed antiepileptic drugs in the world. Despite its pharmacological importance, it may cause liver toxicity and steatosis through mitochondrial ...dysfunction. The aim of this study is to further investigate VPA-induced mechanisms of steatosis by analyzing changes in patterns of methylation in nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Therefore, primary human hepatocytes (PHHs) were exposed to an incubation concentration of VPA that was shown to cause steatosis without inducing overt cytotoxicity. VPA was administered daily for 5 days, and this was followed by a 3 day washout (WO). Methylated DNA regions (DMRs) were identified by using the methylated DNA immunoprecipitation–sequencing (MeDIP-seq) method. The nDNA DMRs after VPA treatment could indeed be classified into oxidative stress- and steatosis-related pathways. In particular, networks of the steatosis-related gene EP300 provided novel insight into the mechanisms of toxicity induced by VPA treatment. Furthermore, we suggest that VPA induces a crosstalk between nDNA hypermethylation and mtDNA hypomethylation that plays a role in oxidative stress and steatosis development. Although most VPA-induced methylation patterns appeared reversible upon terminating VPA treatment, 31 nDNA DMRs (including 5 zinc finger protein genes) remained persistent after the WO period. Overall, we have shown that MeDIP-seq analysis is highly informative in disclosing novel mechanisms of VPA-induced toxicity in PHHs. Our results thus provide a prototype for the novel generation of interesting methylation biomarkers for repeated dose liver toxicity in vitro.
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IJS, KILJ, NUK, PNG, UL, UM
Valproic acid (VPA) is a very potent anti-cancer and neuro-protective drug probably by its HDAC inhibiting properties, which may cause steatosis in the liver. The present study investigates the ...effect of repetitive VPA treatment of primary human hepatocytes (PHH) on whole genome gene expression-, DNA methylation-, and miRNA changes, using microarrays and integrated data analyses. PHH were exposed to a non-cytotoxic dose of VPA for 5days daily which induced lipid accumulation. Part of the PHH was left untreated for 3days for studying the persistence of ‘omics’ changes. VPA treatment appeared to inhibit the expression of the transcription factors HNF1A and ONECUT1. HNF1A interacted with 41 differentially expressed genes of which 12 were also differentially methylated. None of the genes present in this network were regulated by a DE-miR. The subnetwork of ONECUT1 consisted of 44 differentially expressed genes of which 15 were differentially methylated, and 3 were regulated by a DE-miR. A number of genes in the networks are involved in fatty acid metabolism, and may contribute to the development of steatosis by increasing oxidative stress thereby causing mitochondrial dysfunction, and by shifting metabolism of VPA towards β-oxidation due to reduced glucuronidation. Part of the changes remained persistent after washing out of VPA, like PMAIP1 which is associated with cellular stress in liver of patients with NASH. The MMP2 gene showed the highest number of interactions with other persistently expressed genes, among which LCN2 which is a key modulator of lipid homeostasis. Furthermore, VPA modulated the expression and DNA methylation level of nuclear receptors and their target genes involved in the adverse outcome pathway of steatosis, thereby expanding our current knowledge of the pathway. In particular, VPA modulated PPARγ, and PPARα, AHR and CD36 on both the gene expression and the DNA methylation level, thereby inhibiting β-oxidation and increasing uptake of fatty acid into the hepatocytes, respectively. Overall, our integrative data analyses identified novel genes modulated by VPA, which provide more insight into the mechanisms of repeated dose toxicity of VPA, leading to steatosis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Cyclosporine A (CsA) is an undecapeptide with strong immunosuppressant activities and is used a lot after organ transplantation. Furthermore, it may induce cholestasis in the liver. In general, the ...drug-induced cholestasis (DIC) pathway includes genes involved in the uptake, synthesis, conjugation, and secretion of bile acids. However, whether CsA-induced changes in the cholestasis pathway in vitro are persistent for repeated dose toxicity has not yet been investigated. To explore this, primary human hepatocytes (PHH) were exposed to a subcytotoxic dose of 30 μM CsA daily for 3 and 5 days. To investigate the persistence of induced changes upon terminating CsA exposure after 5 days, a subset of PHH was subjected to a washout period (WO-period) of 3 days. Multiple -omics analyses, comprising whole genome analysis of DNA methylation, gene expression, and microRNA expression, were performed. The CsA-treatment resulted after 3 and 5 days, respectively, in 476 and 20 differentially methylated genes (DMGs), 1353 and 1481 differentially expressed genes (DEGs), and in 22 and 29 differentially expressed microRNAs (DE-miRs). Cholestasis-related pathways appeared induced during CsA-treatment. Interestingly, 828 persistent DEGs and 6 persistent DE-miRs but no persistent DMGs were found after the WO-period. These persistent DEGs and DE-miRs showed concordance for 22 genes. Furthermore, 29 persistent DEGs changed into the same direction as observed in livers from cholestasis patients. None of those 29 DEGs which among others relate to oxidative stress and lipid metabolism are yet present in the DIC pathway or cholestasis adverse outcome pathway (AOP) thus presenting novel findings. In summary, we have demonstrated for the first time a persistent impact of repeated dose administration of CsA on genes and microRNAs related to DIC in the gold standard human liver in vitro model with PHH.
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IJS, KILJ, NUK, PNG, UL, UM
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