The nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1), which was initially known to be highly activated by oxidative stress-induced DNA strand breaks, has been shown to be involved in the pathophysiology of acute and chronic inflammatory diseases. PARP-1 deficiency in mice led to the discovery of its coactivating function in the nuclear factorkappa B-mediated gene expression and in addition, pharmaceutical inhibition of PARP-1 was shown to reduce the production of inflammatory mediators. In this study, the in vitro PARP-1-inhibiting effect of various flavonoids was investigated. The flavonoids myricetin, tricetin, gossypetin, delphinidin, quercetin, and fisetin were identified as significant inhibitors of the purified enzyme. Further evaluation of these compounds in N-methyl-N'-nitro-N-nitrosoguanidine-treated human pulmonary epithelial cells showed that the formation of the poly(ADP-ribose) polymers, as well as the decreased NAD 1 levels, was reduced by quercetin, fisetin, and tricetin. Finally, IL-8 production of LPS-stimulated human pulmonary epithelial cells could be significantly reduced by these flavonoids. The results of this study indicate that specific flavonoids have PARP-1-inhibiting activity in addition to the earlier described antioxidant effects. PARP-1 inhibition and preservation of cellular NAD 1 and energy production could play a role in the antiinflammatory activity of these specific flavonoids.In addition, these results indicate additional mechanisms by which flavonoids can exert antiinflammatory activity.Furthermore, these results indicate possibilities to use food-derived flavonoids in the treatment of chronic inflammatory diseases.

There is an increasing need for simple and reliable approaches to phenotypically assess DNA repair capacities. Therefore, a modification of the alkaline comet assay was developed to determine the ability of human lymphocyte extracts to perform the initial steps of the nucleotide excision repair (NER) process, i.e. damage recognition and incision. Gel-embedded nucleoids from A549 cells, pre-exposed to 1 microM benzo[a]pyrene-diol-epoxide, were incubated with cell extracts from frozen or freshly isolated lymphocytes. The rate at which incisions are introduced and the subsequent increase in tail moment is indicative for the repair capacity of the extracts. Freshly prepared extracts from lymphocytes of human volunteers (n = 8) showed significant inter-individual variations in their DNA repair capacity, which correlated with the removal of bulky DNA lesions over a period of 48 h determined by (32)P-post-labelling (R(2) = 0.76, P = 0.005). Repeated measurements revealed a low inter-assay variation (11%). Storage of cell extracts for more than 3 weeks significantly reduced (up to 80%) the capacity to incise the damaged DNA as compared to freshly isolated extracts. This reduction was completely restored by addition of ATP to the extracts before use, as it is required for the incision step of NER. In contrast, extracts freshly prepared from frozen lymphocyte pellets can be used without loss of repair activity. DNA repair deficient XPA-/- and XPC-/- fibroblasts were used to further validate the assay. Although some residual capacity to incise the DNA was observed in these cells, the repair activity was restored to normal wild-type levels when a complementary mixture of both extracts (thereby restoring XPA and XPC deficiency) was used. These results demonstrate that this repair assay can be applied in molecular epidemiological studies to assess inter-individual differences in NER.

Chemical carcinogens may cause a multitude of effects inside cells, thereby affecting transcript levels of genes by direct activation of transcription factors (TF) or indirectly through the formation of DNA damage. As the temporal profiles of these responses may be profoundly different, examining time-dependent changes may provide new insights in TF networks related to cellular responses to chemical carcinogens. Therefore, we investigated in human hepatoma cells gene expression changes caused by benzo[a]pyrene at 12 time points after exposure, in relation to DNA adduct and cell cycle. Temporal profiles for functional gene sets demonstrate both early and late effects in up- and downregulation of relevant gene sets involved in cell cycle, apoptosis, DNA repair, and metabolism of amino acids and lipids. Many significant transcription regulation networks appeared to be around TF that are proto-oncogenes or tumor suppressor genes. The time series analysis tool Short Time-series Expression Miner (STEM) was used to identify time-dependent correlation of pathways, gene sets, TF networks, and biological parameters. Most correlations are with DNA adduct levels, which is an early response, and less with the later responses on G1 and S phase cells. The majority of the modulated genes in the Reactome pathways can be regulated by several of these TF, e.g., 73% by nuclear factor-kappa B and 34-42% by c-MYC, SRF, AP1, and E2F1. All these TF can also regulate one or more of the others. Our data indicate that a complex network of a few TF is responsible for the majority of the transcriptional changes induced by BaP. This network hardly changes over time, despite that the transcriptional profiles clearly alter, suggesting that also other regulatory mechanisms are involved.

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.

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ABSTRACT:Primary human and rat hepatocyte cultures are well established in vitro systems used in toxicological studies. However, whereas transgenic mouse models provide an opportunity for studying mechanisms of toxicity, mouse primary hepatocyte cultures are less well described. The potential usefulness of a mouse hepatocyte-based in vitro model was assessed in this study by investigating time-dependent competence for xenobiotic metabolism and gene expression profiles. Primary mouse hepatocytes, isolated using two-step collagenase perfusion, were cultured in a collagen sandwich configuration. Gene expression profiles and the activities of various cytochrome P450 (P450) enzymes were determined after 0, 42, and 90 h in culture. Principal component analysis of gene expression profiles shows that replicates per time point are similar. Gene expression levels of most phase I biotransformation enzymes decrease to approximately 69 and 57% of the original levels at 42 and 90 h, respectively, whereas enzyme activities for most of the studied P450s decrease to 59 and 34%. The decrease for phase II gene expression is only to 96 and 92% of the original levels at 42 and 90 h, respectively. Pathway analysis reveals initial effects at the level of proteins, external signaling pathways, and energy production. Later effects are observed for transcription, translation, membranes, and cell cycle-related gene sets. These results indicate that the sandwich-cultured primary mouse hepatocyte system is robust and seems to maintain its metabolic competence better than that of the rat hepatocyte system.

The murine embryonic stem cell test (EST) is an alternative testing method designed to assess potential developmental toxicity of compounds. The implementation of transcriptomics in the EST has been shown to reduce the culture duration and improve endpoint evaluation and is expected to result in an enhanced predictability and definition of the applicability domain. We evaluated the identification of developmental toxicity in the EST using two gene sets ("Van_Dartel_heartdiff_24h" and "EST biomarker genes") defined in our earlier studies. Nonexposed embryonic stem cells (ESC) differentiation cultures were sampled 0, 24, and 48 h after initiation of differentiation. Additionally, cultures exposed to 12 diverse well-characterized positive and negative developmental toxicants were isolated 24 h after the onset of exposure. Inhibition of ESC differentiation was evaluated in parallel by morphological scoring on culture day 10. Transcriptomics analysis was conducted using the Affymetrix Gene Chips platform. We applied principal component analysis on the basis of the two predefined gene sets to define the "differentiation track" that represents ESC differentiation. The significance of derivations in the gene expression-based differentiation track because of compound exposures were evaluated to determine developmental toxicity of tested compounds. We successfully predicted developmental toxicity using transcriptomics for 83% (10/12) and 67% (8/12) of the compounds, respectively, using the two predefined gene sets ("Van_Dartel_heartdiff_24h" and "EST biomarker genes"). Our study suggests that the application of transcriptomics may improve the applicability of the EST for the prediction of the developmental toxicity of chemicals.

With the number of new drug candidates increasing every year, there is a need for high-throughput human toxicity screenings. As the liver is the most important organ in drug metabolism and thus capable of generating relatively high levels of toxic metabolites, it is important to find a reliable strategy to screen for drug-induced hepatotoxicity. Microarray-based transcriptomics is a well-established technique in toxicogenomics research and is an ideal approach to screen for drug-induced injury at an early stage. The aim of this study was to prove the principle of classifying known hepatotoxicants and nonhepatotoxicants using their distinctive gene expression profiles in vitro in HepG2 cells. Furthermore, we undertook to subclassify the hepatotoxic compounds by investigating the subclass of cholestatic compounds. Prediction analysis for microarrays was used for classification of hepatotoxicants and nonhepatotoxicants, which resulted in an accuracy of 92% on the training set and 91% on the validation set, using 36 genes. A second model was set up with the goal of finding classifiers for cholestasis, resulting in 12 genes that appeared capable of correctly classifying 8 of the 9 cholestatic compounds, resulting in an accuracy of 93%. We were able to prove the principle that transcriptomic analyses of HepG2 cells can indeed be used to classify chemical entities for hepatotoxicity. Genes selected for classification of hepatotoxicity and cholestasis indicate that endoplasmic reticulum stress and the unfolded protein response may be important cellular effects of drug-induced liver injury. However, the number of compounds in both the training set and the validation set should be increased to improve the reliability of the prediction.