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Background: One of the potential mechanisms linking air pollution to health effects is through changes in DNA-methylation, which so far has mainly been analyzed globally or at candidate sites. Objective: We investigated the association of personal and ambient air pollution exposure measures with genome-wide DNA-methylation changes. Methods: We collected repeated 24-hour personal and ambient exposure measurements of particulate matter (PM2.5), PM2.5 absorbance, and ultrafine particles (UFP) and peripheral blood samples from a panel of 157 healthy non-smoking adults living in four European countries. We applied univariate mixed-effects models to investigate the association between air pollution and genome-wide DNA-methylation perturbations at single CpG (cytosine-guanine dinucleotide) sites and in Differentially Methylated Regions (DMRs). Subsequently, we explored the association of air pollution-induced methylation alterations with gene expression and serum immune marker levels measured in the same subjects. Results: Personal exposure to PM2.5 was associated with methylation changes at 13 CpG sites and 69 DMRs. Two of the 13 identified CpG sites (mapped to genes KNDC1 and FAM50B) were located within these DMRs. In addition, 42 DMRs were associated with personal PM2.5 absorbance exposure, 16 DMRs with personal exposure to UFP, 4 DMRs with ambient exposure to PM2.5, 16 DMRs with ambient PM2.5 absorbance exposure, and 15 DMRs with ambient UFP exposure. Correlation between methylation levels at identified CpG sites and gene expression and immune markers was generally moderate. Conclusion: This study provides evidence for an association between 24-hour exposure to air pollution and DNA-methylation at single sites and regional clusters of CpGs. Analysis of differentially methylated regions provides a promising avenue to further explore the subtle impact of environmental exposures on DNA-methylation. Keywords: DNA-methylation, Air pollution, Fine particles, Ultrafine particles, Immune markers

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 5 days daily which induced lipid accumulation. Part of the PHH was left untreated for 3 days for studying the persistence of 'omics' changes. VPA treatment appeared to inhibit the expression of the transcription factors HNF1A and ONECUTI. 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 ONECUTI 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 beta-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 PPARy, and PPARa, AHR and CD36 on both the gene expression and the DNA methylation level, thereby inhibiting beta-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.

The mechanisms of idiosyncratic drug-induced hepatotoxicity remain largely unclear. It has demonstrated that the drug idiosyncrasy is potentiated in the context of inflammation and intracellular ceramides may play a role in this process.To study the mechanisms, HepG2 cells were co-treated with high and low doses of three idiosyncratic (I) and three non-idiosyncratic (N) compounds, with (I+ and N+) or without (I- and N-) a cytokine mix. Microarray, lipidomics and flow cytometry were performed to investigate the genome-wide expression patterns, the intracellular ceramide levels and the induction of apoptosis.We found that all I + treatments significantly influenced the immune response- and response to stimulus-associated gene ontology (GO) terms, but the induction of apoptotic pathways, which was confirmed by flow cytometry, only appeared to be induced after the high-dose treatment. The ceramide signaling-, ER stress-, NF-kB activation- and mitochondrial activity-related pathways were biologically involved in apoptosis induced by the high-dose I+. Additionally, genes participating in ceramide metabolism were significantly altered resulting in a measurable increase in ceramide levels. The increases in ceramide concentrations may induce ER stress and activate the JNK pathway by affecting the expression of the related genes, and eventually trigger the mitochondria independent apoptosis in hepatocytes. Overall, our study provides a potential mechanism to explain the role of inflammation in idiosyncratic drug reactions. (C) 2017 Elsevier Inc. All rights reserved.

Polychlorinated biphenyls (PCBs) are a class of widespread environmental pollutants, commonly found in human blood, that have been suggested to be linked to the occurrence of sporadic Parkinson's disease (PD). It has been reported that some non-coplanar PCBs accumulate in the brains of female PD patients. To improve our understanding of the association between PCB exposure and PD risk we have applied whole transcriptome gene expression analysis in blood cells from 594 PCB-exposed subjects (369 female, 225 male).Interestingly, we observe that in females, blood levels of non-coplanar PCBs appear to be associated with expression levels of PD-specific genes. However, no such association was detected in males. Among the 131 PD-specific genes affected, 39 have been shown to display similar changes in expression levels in the substantia nigra of deceased PD patients. Especially among the down-regulated genes, transcripts of genes involved in neurotransmitter vesicle-related functions were predominant. (C) 2019 Elsevier Ltd. All rights reserved.

Efforts are put into developing toxicogenomics-based toxicity testing methods usingin vitro human cell models for improving human risk assessment/replacing animal models. Human in vitro liver models include HepG2, HepaRG and primary human hepatocytes (PHH). Studies on comparability/applicability of these cell types mainly focus on assessing baseline biotransformation capacities/cytochrome P450-inducibility, but compound-induced gene expression profiles are at least as important. Therefore, we compared baseline and aflatoxin B1- and benzo(alpha)pyrene-induced gene expression profiles in HepG2, HepaRG and PHH (11-13 donors). At baseline, all liver models differ from each other with respect to whole genome gene expression levels. PHH show profound inter-individual differences, and are most similar to HepaRG. After compound exposure, induced gene expression profiles are more similar between cell models, especially for benzo(a)pyrene. Pathways involved in compound metabolism are induced in all 3 models, while others are more pronounced in a specific cell model. Examples are transcriptomic modifications of carbohydrate-related genes (HepaRG) and of receptor-related genes (PHH) after benzo(alpha)pyrene exposure, and of cell cycle-related genes (HepG2) after aflatoxin B1 exposure. PHH gene expression responses are the most heterogeneous. In conclusion, at base line level PHH are more similar to HepaRG than to HepG2, but for toxicogenomics applications both cell lines perform equally well in comparison to PHH.

Endogenous formation of N-nitroso compounds (NOCs), which are known animal carcinogens, could contribute to human carcinogenesis but definitive evidence is still lacking. To investigate the relevance of NOCs in human colorectal cancer (CRC) development, we analyzed whole genome gene expression modifications in human colon biopsies in relation to fecal NOC exposure. We had a particular interest in patients suffering from intestinal inflammation as this may stimulate endogenous NOC formation, and consequently predispose to CRC risk. Inflammatory bowel disease (IBD) patients diagnosed with ulcerative colitis and irritable bowel syndrome patients without inflammation, serving as controls, were therefore recruited. Fecal NOC were demonstrated in the majority of subjects. By associating gene expression levels of all subjects to fecal NOC levels, we identified a NOC exposure-associated transcriptomic response that suggests that physiological NOC concentrations may potentially induce genotoxic responses and chromatin modifications in human colon tissue, both of which are linked to carcinogenicity. In a network analysis, chromatin modifications were linked to 11 significantly modulated histone genes, pointing towards a possible epigenetic mechanism that may be relevant in comprehending NOC-induced carcinogenesis. In addition, pro-inflammatory transcriptomic modifications were identified in visually non-inflamed regions of the IBD colon. However, fecal NOC levels were slightly but not significantly increased in IBD patients, suggesting that inflammation did not strongly stimulate NOC formation. We conclude that NOC exposure is associated with gene expression modifications in the human colon that may suggest a potential role of these compounds in CRC development.

A compound for which marked species differences have been reported in laboratory animals and humans is coumarin. In rats, metabolites of coumarin are highly toxic, whereas in humans, the compound is mainly metabolized to non-toxic metabolites. In the present study, a toxicogenomics-based parallelogram approach was used to compare effects of coumarin on gene expression in human hepatocytes relevant for the situation in vivo. To this purpose, gene expression profiling was performed on human hepatocytes treated with coumarin in a pharmacological relevant and proposed toxic concentration and results were compared to a previously performed coumarin in vivo and in vitro rat toxicogenomics study. No cytotoxicity was observed in human hepatocytes at both concentrations, whereas rats showed clear toxic effects in vitro as well as in vivo. In all three systems, coumarin affected genes involved in the blood coagulation pathway; this indicates relevant responses in cases of human exposure. However, no pathways and processes related to hepatotoxicity in rats were observed in human hepatocytes. Still, repression of energy-consuming biochemical pathways and impairment of mitochondrial function were observed in human hepatocytes treated with the highest concentration of coumarin, possibly indicating toxicity. In conclusion, although species differences in response to coumarin are evident in the present results, the toxicogenomics-based parallelogram approach enables clear discrimination between pharmacological responses at pharmacological doses and proposed toxic responses at high (toxic) doses relevant for humans in vivo.

Antioxidants are of major importance in the protection against cellular oxidative damage caused by endogenous as well as exogenous free radicals. This study aims to establish the impact of genetic polymorphisms in GSTM1 and GSTT1 , which encode for enzymatic antioxidative defence, on H 2 O 2 -induced oxidative DNA damage and on the effectiveness of quercetin and ascorbic acid in preventing this induced damage in human lymphocytes. Lymphocytes from 12 healthy volunteers were pre-incubated either with 10 μM of quercetin or with 10 μM of ascorbic acid, and exposed to 25 μM H 2 O 2 for 1 h. The induction of oxidative DNA damage was quantified using the Comet assay. Genotyping of these 12 subjects showed that six individuals were GSTM1+ and six were GSTM1− ; eight were GSTT1+ and four GSTT1− . Results Baseline levels of oxidative DNA damage did not differ between GSTM1 or GSTT1 variants and their respective wild types. Also with respect to ex vivo induced levels of oxidative DNA damage, no significant difference was seen between variants and wild types of both genotypes. The protection against H 2 O 2 -induced oxidative DNA damage by quercetin was significantly higher in GSTT1 wild types than in GSTT1 variants (57% and 9% decrease, respectively; p = 0.01); furthermore, GSTT1 wild types were protected against induced oxidative DNA damage by ascorbic acid pre-incubation while GSTT1 variants showed an increase of damage (16% decrease vs. 91% increase; p = 0.01). For GSTM1 variants and wild types, observed differences in protective effects of quercetin or ascorbic acid were not statistically significant. Overall, quercetin proves to be better in protecting human lymphocytes in vitro against oxidative DNA damage upon H 2 O 2 challenge than ascorbic acid.

A rapid decline of cytochrome P450 (CYP450) enzyme activities remains a drawback of rat hepatocyte-based in vitro cultures. Consequently, judgment of the toxic potential of compounds that need bioactivation by CYP450s may not be adequate using this model. In the present study, an improved hepatocyte-based in vitro system was developed with special focus on metabolic competence. Therefore, a mixture of CYP450 inducers, phenobarbital, dexamethasone and beta-naphthoflavone, was added to culture medium of sandwich-cultured rat hepatocytes. The resulting modified model was evaluated by comparing its genome-wide expression profiles with liver and a standard model without the inducer mixture. Metabolic capacity for CYP450 enzymes showed that the modified model resembled more closely the in vivo situation. Gene expression results revealed large differences between in vivo and both in vitro models. The slight differences between the two sandwich models were predominantly represented by gene expression changes in CYP450s. Importantly, in the modified model, expression ratios of the phase I and the majority of phase II genes more closely resembled liver in vivo. The CYP450 enzyme activities corresponded with gene expression data. In conclusion, for toxicological applications using sandwich-cultured hepatocytes, the modified model may be preferred. AD - Business Unit Biosciences, TNO Quality of Life, P.O. Box 360, 3700 AJ, Zeist, The Netherlands; Department of Health Risk Analysis and Toxicology, University of Maastricht, Maastricht, The Netherlands.

Colorectal cancer (CRC) is believed to be related to the intake of processed meat and the formed heterocyclic aromatic amines (HCA) herein, which are metabolically activated by the enzymes cytochrome P4501A2 (CYP1A2) and N-acetyltransferase 2 (NAT2). The influence of genotypic and phenotypic variations for CYP1A2 and NAT2 on the risk for colorectal adenomas was investigated in 94 individuals at different risk of developing CRC. Significant associations were found between the CYP1A2-164A-->C polymorphism (CYP1A2*1F) and the risk of colorectal adenomas, suggesting that the studied polymorphism plays an important role in CRC risk in humans.

We present Sr, C and O isotope ratios and quantitative ratios of trace element ratios in the rib and the femur of the Tyrolean Iceman “Otzi”. The results from the rib and the femur are compared with those obtained from pre-industrial historic human skull bone fragments collected in the late 19th century from charnel houses located on different geological backgrounds throughout North and South Tyrol. Comparison of strontium isotope data from the bone fragments locates the Iceman's domicile, during the last 5 to 10 years of his life, to the crystalline part of the Central Alps. Statistical analysis of the trace element ratios, together with oxygen isotope results, suggests that the Iceman probably spent most of his last years in southern elevated part of the Otztal. However, the results from Iceman only represent those from a single individual and are not a representative average of a group. It cannot therefore be established with absolute certainty that the original trace element signatures were conserved, especially during the first stage of mummification. Our conclusions should therefore be regarded as tentative, but nevertheless the best possible indication his domicile, and a stimulus for further research.

Department of Health Risk Analysis and Toxicology, Maastricht University, Netherlands.Various markers of radiation-induced DNA damage including DNA oxidation were investigated in peripheral lymphocytes of 23 cancer patients prior to and one week after receiving radiotherapy with a cumulative dose of 54-70 Gy. Exposure to ionizing radiation nonsignificantly increased the ratio 2'deoxy-7-dihydro-8-oxoguanosine/2'deoxyguanosine (8-oxodG/dG) from 1.73 x 10(-5) to 3.33 x 10(-5). Frequencies of micronuclei significantly (p = 0.0003) increased from 6.4 to 38.9 per 1000 cells. The frequency of hypoxanthine-guanine-phosphoribosyltransferase (HPRT) mutant lymphocytes measured as 6-thioguanine resistant variant cells by 5-bromodeoxyuridine labeling, was elevated eight-fold, from 4.7 x 10(-6) to 36.2 x 10(-6) (p = 0.008) after termination of the radiotherapy, thus showing a clear response to the radiation treatment. No correlation between levels of oxidative DNA damage and frequencies of HPRT mutant lymphocytes or micronuclei could be established.

DNA adduct analysis is often used for biomonitoring individuals exposed to polycyclic aromatic hydrocarbons (PAH). The 32P-postlabeling assay is routinely applied to study the formation of aromatic bulky adducts, but cannot positively identify individual adduct types. Recently, an HPLC assay with fluorescence detection (HPLC-FD) was developed which was sufficiently sensitive to detect adducts formed by benzo[a]pyrene (B[a]P) diolepoxide isomers [(+/-)anti- and (+/-)syn-BPDE] in occupationally exposed subjects (Rojas et al. Carcinogenesis, 16 (1995) 1373-1376). In this study, we compared both techniques using DNA samples of rats which were treated i.p. with B[a]P (10 mg/kg bw). The internal dose was assessed by measuring 3-OH-B[a]P excretion in urine. The detection limit of the HPLC-FD assay varied from 0.5 to 7.4 adducts per 10(8) nucleotides, while the detection limit of the 32P-postlabeling assay was around 1 adduct per 10(9) nucleotides. HPLC-FD analysis showed that BPDE-DNA adduct levels were highest in the heart, lung and liver respectively. The most predominant B[a]P-tetrol was the I-1 isomer, which derives from hydrolysis of the major reaction product of DNA and (+)-anti-BPDE. 32P-postlabeling analysis revealed an adduct spot that comigrated with a [3H]BPDE-DNA standard. The putative BPDE-DNA adduct levels were highest in heart followed by lung and liver and correlated significantly with tetrol I-1 levels determined by HPLC-FD (r = 0.72, P = 0.006). In samples in which both tetrol I-1 and II-2 were detected by means of HPLC-FD, this correlation was even better (r = 0.95, P = 0.01). Estimated half-lives of BPDE-DNA adducts were in the ranking order; heart, lung and liver for both techniques. By 32P-postlabeling, adducts other than BPDE-DNA were also found, resulting in highest total DNA adduct levels in the liver, heart and lung respectively. Furthermore, mean 24 h urinary excretion of 3-OH-B[a]P was related to BPDE-DNA adduct levels in lung, liver and heart. The 32P-postlabeling assay is sensitive and capable of detecting exposures to complex mixtures, whereas the HPLC-FD assay can be used to identify BPDE-isomers and might therefore be of value in risk assessment of individuals exposed to PAH.

Butylated hydroxyanisole (BHA) is a synthetic food antioxidant used to prevent oils, fats and shortenings from oxidative deterioration and rancidity. This review depicts the current knowledge on BHA. The physical and chemical characteristics of BHA are summarized and its function as a food antioxidant is made clear. The toxicological characteristics of BHA and its metabolic fate in man and animal are briefly reviewed. Special emphasis is laid on the carcinogenicity of BHA in the forestomach of rodents and to related events in the forestomach and other tissues in experimental animals. At present there is sufficient evidence for carcinogenicity of BHA, but there is hardly any indication that BHA is genotoxic. Therefore risk assessment for this epigenetic carcinogen is based on non-stochastic principles. However, the mechanism underlying the tumorigenicity of BHA is not known. In the last part of this review an attempt is made to unravel the unknown mechanism of carcinogenicity. It is hypothesized that BHA gives rise to tumor formation in rodent forestomach by inducing heritable changes in DNA. Evidence is being provided that reactive oxygen species, in particular hydroxylradicals, may play a crucial role. The key question with respect to risk assessment for BHA is whether or not the underlying mechanism is thresholded, which is important for the choice of the appropriate model to assess the risk, if any, for man and to manage any potential risk.

Journal URL: http://www.sciencedirect.com/science/journal/13835718

Naturally occurring fecal mutagens, called fecapentaenes, are hypothesized to contribute to large bowel carcinogenesis. To understand health risks it is necessary to relate fecal mutagen concentrations to intestinal pathologies, i.e. colon cancer. However, fast and reliable methods for analysis of stool for fecapentaene levels are not available. This study presents an evaluation of stabilizing effects on synthetic fecapentaene-12 of various antioxidants, indicating tri-ethylamine to give the best results. Furthermore, it describes a fast extraction procedure for human stool and a subsequent HPLC-analysis which produces quantitative data on fecapentaene-12 concentrations within 30 min.

Mutagenic activity of repeatedly used deep-frying fats was evaluated in relation to chemical characteristics. Deep-frying fat samples were collected from local restaurants and snack bars after sensory indication of abuse. A total of 20 deep-frying fat samples and 2 unused control fat samples was tested. Fat samples were fractionated into non-polar and polar compounds by column chromatography. Amounts of polar compounds obtained ranged from 2% (by weight) for unused fat to 44% for used deep-frying fat. Levels of di- and poly-meric triglycerides (DPTG) were determined using gel-permeation chromatography. DPTG concentrations of 13 used deep-frying fat samples exceeded the treshold level of 10% above which fats are rejected for use. In addition thiobarbituric acid-reactive substances (TBA-RS) were measured. Amounts of TBA-RS were just above detection levels for most fat samples. Five used fat samples, however, contained relatively high concentrations of TBA-RS, ranging from 82 to 177 moles malondialdehyde/g. Non-polar and polar fractions were screened for mutagenic activity using the Ames mutagenicity assay. Mutagenic activity was found predominantly in polar fractions at doses higher than 1 mg/plate in strains TA97, TA100 and TA104, variously with and without metabolic activation. The highest number of mutagenic samples was detected by strain TA97, which appeared to be most sensitive. Some samples exhibited toxic effects. Chromatography blanks, consisting of solvents processed according to the same procedures as used for fat samples, were not mutagenic. Mutagenic activity was also detected in polar material obtained from unused frying fat. Non-polar fractions of unused frying fats showed no mutagenicity. A frying experiment carried out under laboratory conditions indicated that during repeated and prolonged use of deep-frying fat mutagenic polar substances were formed. Fat samples taken after 20 and 40 h of frying contained increasing amounts of polar compounds. Mutagenic activity was highest after 20 h of frying but was slightly decreased after 40 h of frying. At this stage, however, mutagens also appeared in the non-polar fraction. Mutagenic activity of polar fractions of used deep-frying fats in strain TA97 was positively correlated with levels of TBA-RS, which may indicate the involvement of lipid oxidation products in mutagenicity of used deep-frying fats. No significant correlations were found with other chemical characteristics. In the process of deep-fat frying numerous degradation products are formed, which may include mutagenic heterocyclic amines and other pyrolysates. At present the specific nature of mutagens formed during deep fat frying is unclear.