Recent progress in the genetics and epigenetics of paraoxonase: why it is relevant to children's environmental health.

PURPOSE OF REVIEW: Children are more susceptible to exposures in utero and during early childhood that may result in developmental problems and chronic diseases. Novel discoveries in the field of molecular epidemiology that can help explain susceptibility to exposures and disease will be demonstrated using the multifunctional enzyme paraoxonase 1 (PON1) as an example. RECENT FINDINGS: The broad PON1 variability in humans, partly due to differences in genetics and age, can confer differential susceptibility because this enzyme can detoxify organophosphate pesticides and has antioxidant properties. Epigenetics plays a significant role in the mediation of the effects of environmental exposure on human health and is hypothesized to be a major contributing factor to the early-life origins of adult disease. Studies highlighted in this review demonstrate the relationship of PON1 polymorphisms with microRNA binding in addition to a link between DNA methylation in the transcriptional regulatory region with changes in PON1 enzyme levels. Other important methodologies such as ancestry informative markers and lactonase activity can enhance studies involving PON1. SUMMARY: This PON1 model demonstrates that integrating genetic and epigenetic factors, as well as other novel methodologies, can improve our understanding of important susceptibility factors linked to pediatric disease.

Characterisation of cisplatin-induced transcriptomics responses in primary mouse hepatocytes, HepG2 cells and mouse embryonic stem cells shows conservation of regulating transcription factor networks.

The toxic mechanisms of cisplatin have been frequently studied in many species and in vitro cell models. The Netherlands Toxicogenomics Centre focuses on developing in vitro alternatives using genomics technologies for animal-based assays on, e.g. genotoxic hazards. Models such as human hepatocellular carcinoma cell line (HepG2) cells, mouse primary hepatocytes (PMH) and mouse embryonic stem cells (mESC) are used. Our aim was to identify possibly robust conserved mechanisms between these models using cisplatin as model genotoxic agent. Transcriptomic data newly generated from HepG2 cells and PMH exposed to 7 µM cisplatin for 12, 24 and 48h and 24 and 48h, respectively, were compared with published data from mESC exposed to 5 µM cisplatin for 2-24h. Due to differences in response time between models and marginal changes after shorter exposure periods, we focused on 24 and 48h. At gene level, 44 conserved differentially expressed genes (DEG), involved in processes such as apoptosis, cell cycle, DNA damage response and DNA repair, were found. Functional analysis shows that limited numbers of pathways are conserved. Transcription factor (TF) network analysis indicates 12 common TF networks responding among all models and time points. Four TF, HNF4-α, SP1, c-MYC and p53, capable of regulating ±50% of all DEG, seem of equal importance in all models and exposure periods. Here we showed that transcriptomic responses across several in vitro cell models following exposure to cisplatin are mainly determined by a conserved complex network of 4 TFs. These conserved responses are hypothesised to provide most relevant information for human toxicity prediction and may form the basis for new in vitro alternatives of risk assessment.

Grape antioxidant dietary fiber inhibits intestinal polyposis in ApcMin/+ mice: relation to cell cycle and immune response.

Epidemiological and experimental studies suggest that fiber and phenolic compounds might have a protective effect on the development of colon cancer in humans. Accordingly, we assessed the chemopreventive efficacy and associated mechanisms of action of a lyophilized red grape pomace containing proanthocyanidin (PA)-rich dietary fiber [grape antioxidant dietary fiber (GADF)] on spontaneous intestinal tumorigenesis in the Apc(Min/+) mouse model. Mice were fed a standard diet (control group) or a 1% (w/w) GADF-supplemented diet (GADF group) for 6 weeks. GADF supplementation greatly reduced intestinal tumorigenesis, significantly decreasing the total number of polyps by 76%. Moreover, size distribution analysis showed a considerable reduction in all polyp size categories [diameter <1mm (65%), 1-2mm (67%) and >2mm (87%)]. In terms of polyp formation in the proximal, middle and distal portions of the small intestine, a decrease of 76, 81 and 73% was observed, respectively. Putative molecular mechanisms underlying the inhibition of intestinal tumorigenesis were investigated by comparison of microarray expression profiles of GADF-treated and non-treated mice. We observed that the effects of GADF are mainly associated with the induction of a G1 cell cycle arrest and the downregulation of genes related to the immune response and inflammation. Our findings show for the first time the efficacy and associated mechanisms of action of GADF against intestinal tumorigenesis in Apc(Min/+) mice, suggesting its potential for the prevention of colorectal cancer.

Benzo[a]pyrene-induced changes in microRNA-mRNA networks.

Toxicological studies assessing the safety of compounds for humans frequently use in vitro systems to characterize toxic responses in combination with transcriptomic analyses. Thus far, changes have mostly been investigated at the mRNA level. Recently, microRNAs have attracted attention because they are powerful negative regulators of mRNA levels and, thus, may be responsible for the modulation of important mRNA networks implicated in toxicity. This study aimed to identify possible microRNA-mRNA networks as novel interactions on the gene expression level after a genotoxic insult. We used benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon, as a model genotoxic/carcinogenic compound. We analyzed time-dependent effects on mRNA and microRNA profiles in HepG2 cells, a widely used human liver cell line that expresses active p53 and is competent for the biotransformation of BaP. Changes in microRNA expression in response to BaP, in combination with multiple alterations of mRNA levels, were observed. Many of these altered mRNAs are targets of altered microRNAs. Using pathway analysis, we evaluated the relevance of such microRNA deregulations to genotoxicity. This revealed eight microRNAs that appear to participate in specific BaP-responsive pathways relevant to genotoxicity, such as apoptotic signaling, cell cycle arrest, DNA damage response, and DNA damage repair. Our results particularly highlight the potential of microRNA-29b, microRNA-26a-1*, and microRNA-122* as novel players in the BaP response. Therefore, this study demonstrates the added value of an integrated microRNA-mRNA approach for identifying molecular mechanisms induced by BaP in an in vitro human model.

The natural triterpene maslinic acid induces apoptosis in HT29 colon cancer cells by a JNK-p53-dependent mechanism.

BACKGROUND: Maslinic acid, a pentacyclic triterpene found in the protective wax-like coating of the leaves and fruit of Olea europaea L., is a promising agent for the prevention of colon cancer. We have shown elsewhere that maslinic acid inhibits cell proliferation to a significant extent and activates mitochondrial apoptosis in colon cancer cells. In our latest work we have investigated further this compound's apoptotic molecular mechanism. METHODS: We used HT29 adenocarcinoma cells. Changes genotoxicity were analyzed by single-cell gel electrophoresis (comet assay). The cell cycle was determined by flow cytometry. Finally, changes in protein expression were examined by western blotting. Student's t-test was used for statistical comparison. RESULTS: HT29 cells treated with maslinic acid showed significant increases in genotoxicity and cell-cycle arrest during the G0/G1 phase after 72 hours' treatment and an apoptotic sub-G0/G1 peak after 96 hours. Nevertheless, the molecular mechanism for this cytotoxic effect of maslinic acid has never been properly explored. We show here that the anti-tumoral activity of maslinic acid might proceed via p53-mediated apoptosis by acting upon the main signaling components that lead to an increase in p53 activity and the induction of the rest of the factors that participate in the apoptotic pathway. We found that in HT29 cells maslinic acid activated the expression of c-Jun NH2-terminal kinase (JNK), thus inducing p53. Treatment of tumor cells with maslinic acid also resulted in an increase in the expression of Bid and Bax, repression of Bcl-2, release of cytochrome-c and an increase in the expression of caspases -9, -3, and -7. Moreover, maslinic acid produced belated caspase-8 activity, thus amplifying the initial mitochondrial apoptotic signaling. CONCLUSION: All these results suggest that maslinic acid induces apoptosis in human HT29 colon-cancer cells through the JNK-Bid-mediated mitochondrial apoptotic pathway via the activation of p53. Thus we propose a plausible sequential molecular mechanism for the expression of the different proteins responsible for the intrinsic mitochondrial apoptotic pathway. Further studies with other cell lines will be needed to confirm the general nature of these findings.

A lyophilized red grape pomace containing proanthocyanidin-rich dietary fiber induces genetic and metabolic alterations in colon mucosa of female C57BL/6J mice.

Diet plays a decisive role in promoting or preventing colon cancer. However, the specific effects of some nutrients remain unclear. The capacity of fruit and vegetables to prevent cancer has been associated with their fiber and antioxidant composition. We investigated whether consumption of a lyophilized red grape pomace containing proanthocyanidin-rich dietary fiber (grape antioxidant dietary fiber, GADF) by female C57BL/6J mice would affect the serum metabolic profile or colon mucosa gene expression using NMR techniques and DNA microarray, respectively. The mice were randomly assigned to 2 groups that for 2 wk consumed a standard rodent diet and were gavaged with 100 mg/kg body weight GADF suspended in water or an equivalent volume of plain tap water (10 mL/kg body weight). The amount of fiber supplemented was calculated to equal the current recommended daily levels of fiber consumption for humans. The inclusion of dietary GADF induced alterations in the expression of tumor suppressor genes and proto-oncogenes as well as the modulation of genes from pathways, including lipid biosynthesis, energy metabolism, cell cycle, and apoptosis. Overexpression of enzymes pertaining to the xenobiotic detoxifying system and endogenous antioxidant cell defenses was also observed. In summary, the genetic and metabolic profiles induced by GADF were consistent with the preventive effects of fiber and polyphenols. On the basis of these observations, we propose that GADF may contribute to reducing the risk of colon cancer.

Age-Related Differences in miRNA Expression in Mexican-American Newborns and Children.

Epigenetic mechanisms have emerged as an important pathway through which environmental exposures can affect health through the regulation of gene expression without changes in DNA sequence: microRNAs (miRNAs) are short non-coding RNAs that target protein-coding mRNAs, leading to post-transcriptional repression. They are involved in important physiologic processes, but little is known about how miRNA expression may change with age in children. We used an nCounter miRNA assay to assess the expression of 43 miRNAs in buffy coat samples collected from newborns (n = 121) and 7-year-old (n = 142) children. We identified 36 miRNAs that were differentially expressed between newborns and 7-year-olds after controlling for blood cell composition. Using pathway analysis, we found that differentially expressed miRNAs targeted genes enriched for processes related to post-translational modifications, metabolism, and immune response. Our study found that unlike adults, where miRNA expression levels in peripheral blood may decrease with age, expression levels of most miRNAs increased from birth to mid-childhood. This may be reflective of the role miRNAs may play in the highly coordinated mechanisms regulating genes involved in children's development. Furthermore, it will be important to adjust for both age and blood cell composition in future pediatric studies of miRNA expression in blood.

miRNAs differentially expressed by next-generation sequencing in cord blood buffy coat samples of boys and girls.

AIM: Differences in children's development and susceptibility to diseases and exposures have been observed by sex, yet human studies of sex differences in miRNAs are limited. MATERIALS & METHODS: The genome-wide miRNA expression was characterized by sequencing-based EdgeSeq assay in cord blood buffy coats from 89 newborns, and 564 miRNAs were further analyzed. RESULTS: Differential expression of most miRNAs was higher in boys. Neurodevelopment, RNA metabolism and metabolic ontology terms were enriched among miRNA targets. The majority of upregulated miRNAs (86%) validated by nCounter maintained positive-fold change values; however, only 21% reached statistical significance by false discovery rate. CONCLUSION: Accounting for host factors like sex may improve the sensitivity of epigenetic analyses for epidemiological studies in early childhood.

Procyanidin fractions from pine (Pinus pinaster) bark: radical scavenging power in solution, antioxidant activity in emulsion, and antiproliferative effect in melanoma cells.

Pine (Pinus pinaster) bark is a rich source of procyanidin oligomers. From a total polyphenolic extract, we have generated fractions of different procyanidin composition. The mixtures, devoid of gallate esters, were active as free radical scavengers against ABTS(*+), DPPH, and HNTTM. Pine bark fractions were tested for antioxidant activity in solution (hydrogen donation and electron transfer) and emulsion (inhibition of lipid peroxidation) and compared with their galloylated counterparts from grape origin. While galloylation clearly influenced the free radical scavenging efficiency in solution, it did not seem to play a determinant role in protection against lipid peroxidation in emulsion. The fractions were very mild inhibitors of cell proliferation. Because gallate esters appear to interfere with crucial cell functions, gallate free pine procyanidins may be the innocuous chemopreventative agents of choice for many applications in food and skin protection.

Screening for drug-induced hepatotoxicity in primary mouse hepatocytes using acetaminophen, amiodarone, and cyclosporin a as model compounds: an omics-guided approach.

Drug-induced hepatotoxicity is a leading cause of attrition for candidate pharmaceuticals in development. New preclinical screening methods are crucial to predict drug toxicity prior to human studies. Of all in vitro hepatotoxicity models, primary human hepatocytes are considered as 'the gold standard.' However, their use is hindered by limited availability and inter-individual variation. These barriers may be overcome by using primary mouse hepatocytes. We used differential in gel electrophoresis (DIGE) to study large-scale protein expression of primary mouse hepatocytes. These hepatocytes were exposed to three well-defined hepatotoxicants: acetaminophen, amiodarone, and cyclosporin A. Each hepatotoxicant induces a different hepatotoxic phenotype. Based on the DIGE results, the mRNA expression levels of deregulated proteins from cyclosporin A-treated cells were also analyzed. We were able to distinguish cyclosporin A from controls, as well as acetaminophen and amiodarone-treated samples. Cyclosporin A induced endoplasmic reticulum (ER) stress and altered the ER-Golgi transport. Moreover, liver carboxylesterase and bile salt sulfotransferase were differentially expressed. These proteins were associated with a protective adaptive response against cyclosporin A-induced cholestasis. The results of this study are comparable with effects in HepG2 cells. Therefore, we suggest both models can be used to analyze the cholestatic properties of cyclosporin A. Furthermore, this study showed a conserved response between primary mouse hepatocytes and HepG2 cells. These findings collectively lend support for use of omics strategies in preclinical toxicology, and might inform future efforts to better link preclinical and clinical research in rational drug development.

RNA-Seq provides new insights in the transcriptome responses induced by the carcinogen benzo[a]pyrene.

Whole-genome transcriptome measurements are pivotal for characterizing molecular mechanisms of chemicals and predicting toxic classes, such as genotoxicity and carcinogenicity, from in vitro and in vivo assays. In recent years, deep sequencing technologies have been developed that hold the promise of measuring the transcriptome in a more complete and unbiased manner than DNA microarrays. Here, we applied this RNA-seq technology for the characterization of the transcriptomic responses in HepG2 cells upon exposure to benzo[a]pyrene (BaP), a well-known DNA damaging human carcinogen. Based on EnsEMBL genes, we demonstrate that RNA-seq detects ca 20% more genes than microarray-based technology but almost threefold more significantly differentially expressed genes. Functional enrichment analyses show that RNA-seq yields more insight into the biology and mechanisms related to the toxic effects caused by BaP, i.e., two- to fivefold more affected pathways and biological processes. Additionally, we demonstrate that RNA-seq allows detecting alternative isoform expression in many genes, including regulators of cell death and DNA repair such as TP53, BCL2 and XPA, which are relevant for genotoxic responses. Moreover, potentially novel isoforms were found, such as fragments of known transcripts, transcripts with additional exons, intron retention or exon-skipping events. The biological function(s) of these isoforms remain for the time being unknown. Finally, we demonstrate that RNA-seq enables the investigation of allele-specific gene expression, although no changes could be observed. Our results provide evidence that RNA-seq is a powerful tool for toxicology, which, compared with microarrays, is capable of generating novel and valuable information at the transcriptome level for characterizing deleterious effects caused by chemicals.

Effect of Trichostatin A on miRNA expression in cultures of primary rat hepatocytes.

In the present study, the effect of Trichostatin A (TSA), a histone deacetylase inhibitor, was investigated on the microRNA (miR, miRNA) expression profile in cultured primary rat hepatocytes by means of microarray analysis. Simultaneously, albumin secretory capacity and morphological features of the hepatocytes were evaluated throughout the culture time. In total, 25 out of 348 miRNAs were found to be differentially expressed between freshly isolated hepatocytes and 7-day cultured cells. Nineteen of these miRNAs were connected with 'general metabolism'. miR-21 and miR-126 were shown to be the most up and down regulated miRs upon cultivation and could be linked to the proliferative response triggered in the hepatocytes upon their isolation from the liver. miR-379 and miR-143, on the other hand, were found to be the most up and down regulated miRs upon TSA treatment. Together with the higher expression of miR-122 observed in TSA-treated versus non-treated cultures, we hypothesize that the changes observed for miR-122, miR-143 and miR-379 could be related to the inhibitory effects of TSA on hepatocellular proliferation.

Highly galloylated tannin fractions from witch hazel (Hamamelis virginiana) bark: electron transfer capacity, in vitro antioxidant activity, and effects on skin-related cells.

Witch hazel ( Hammamelis virginiana) bark is a rich source of both condensed and hydrolizable oligomeric tannins. From a polyphenolic extract soluble in both ethyl acetate and water, we have generated fractions rich in pyrogallol-containing polyphenols (proanthocyanidins, gallotannins, and gallates). The mixtures were highly active as free radical scavengers against ABTS, DPPH (hydrogen donation and electron transfer), and HNTTM (electron transfer). They were also able to reduce the newly introduced TNPTM radical, meaning that they included some highly reactive components. Witch hazel phenolics protected red blood cells from free radical-induced hemolysis and were mildly cytotoxic to 3T3 fibroblasts and HaCat keratinocytes. They also inhibited the proliferation of tumoral SK-Mel 28 melanoma cells at lower concentrations than grape and pine procyanidins. The high content in pyrogallol moieties may be behind the effect of witch hazel phenolics on skin cells. Because the most cytotoxic and antiproliferative mixtures were also the most efficient as electron transfer agents, we hypothesize that the final putative antioxidant effect of polyphenols may be in part attributed to the stimulation of defense systems by mild prooxidant challenges provided by reactive oxygen species generated through redox cycling.

Witch hazel (Hamamelis virginiana) fractions and the importance of gallate moieties--electron transfer capacities in their antitumoral properties.

Witch hazel (Hamamelis virginia) extracts are used in traditional medicine. They are particularly rich in gallate esters included in proanthocyanidins, hydrolyzable tannins (galloylated sugars), and methyl gallate. This study examines the response of human colon cancer cells to treatment with fractions obtained from a witch hazel polyphenolic extract. The results are compared with those obtained previously with homologous fractions from grape (less galloylated) and pine (nongalloylated). Witch hazel fractions were the most efficient in inhibiting cell proliferation in HT29 and HCT116 human colon cancer cell lines, which clearly shows that the more galloylated the fractions, the more effective they were at inhibiting proliferation of colon cancer cells. Witch hazel fractions were, in addition, more potent in arresting the cell cycle at the S phase and inducing apoptosis; they also induced a significant percentage of necrosis. Interestingly, the apoptosis and cell cycle arrest effects induced were proportional to their galloylation. Moreover, witch hazel fractions with a high degree of galloylation were also the most effective as scavengers of both hydroxyl and superoxide radicals and in protecting against DNA damage triggered by the hydroxyl radical system. These findings provide a better understanding of the structure-bioactivity relationships of polyphenolics, which should be of assistance in choosing an appropriate source and preparing a rational design for formulations of plant polyphenols in nutritional supplements.