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The EU FP6 project carcinoGENOMICS explored the combination of toxicogenomics and in vitro cell culture models for identifying organotypical genotoxic- and non-genotoxic carcinogen- specific gene signatures. Here the performance of its gene classifier, derived from exposure of metabolically competent human HepaRG cells to prototypical non-carcinogens (10 compounds) and hepatocarcinogens (20 compounds), is reported. Analysis of the data at the gene and the pathway level by using independent biostatistical approaches showed a distinct separation of genotoxic from non-genotoxic hepatocarcinogens and non-carcinogens (up to 88% correct prediction). The most characteristic pathway responding to genotoxic exposure was DNA damage. Interlaboratory reproducibility was assessed by blindly testing of three compounds, from the set of 30 compounds, by three independent laboratories. Subsequent classification of these compounds resulted in correct prediction of the genotoxicants. As expected, results on the non-genotoxic carcinogens and the non-carcinogens were less predictive. In conclusion, the combination of transcriptomics with the HepaRG in vitro cell model provides a potential weight of evidence approach for the evaluation of the genotoxic potential of chemical substances. [ABSTRACT FROM AUTHOR]

The primary cilium is an immotile sensory and signaling organelle found on the majority of mammalian cell types. Of the multitude of roles that the primary cilium performs, perhaps some of the most important include maintenance of differentiation, quiescence, and cellular polarity. Given that the progression of cancer requires disruption of all of these processes, we have investigated the effects of several carcinogens on the primary cilium of the RPTEC/TERT1 human proximal tubular epithelial cell line. Using both scanning electron microscopy and immunofluorescent labeling of the ciliary markers acetylated tubulin and Arl13b, we confirmed that RPTEC/TERT1 cells express primary cilium upon reaching confluence. Treatment with the carcinogens ochratoxin A (OTA) and potassium bromate (KBrO3) caused a significant reduction in the number of ciliated cells, while exposure to nifedipine, a noncarcinogenic renal toxin, had no effect on primary cilium expression. Flow cytometric analysis of the effects of all three compounds on the cell cycle revealed that only KBrO3 resulted in an increase in the proportion of cells entering the cell cycle. Microarray analysis revealed dysregulation of multiple pathways affecting ciliogenesis and ciliary maintenance following OTA and KBrO3 exposure, which were unaffected by nifedipine exposure. The primary cilium represents a unique physical checkpoint with relevance to carcinogenesis. We have shown that the renal carcinogens OTA and KBrO3 cause significant deciliation in a model of the proximal tubule. With KBrO3, this was followed by reentry into the cell cycle; however, deciliation was not found to be associated with reentry into the cell cycle following OTA exposure. Transcriptomic analysis identified dysregulation of Wnt signaling and ciliary trafficking in response to OTA and KBrO3 exposure. [ABSTRACT FROM AUTHOR]

Background: In vitro cell systems together with omics methods represent promising alternatives to conventional animal models for toxicity testing. Transcriptomic and proteomic approaches have been widely applied in vitro but relatively few studies have used metabolomics. Therefore, the goal of the present study was to develop an untargeted methodology for performing reproducible metabolomics on in vitro systems. The human liver cell line HepG2, and the well-known hepatotoxic and non-genotoxic carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), were used as the in vitro model system and model toxicant, respectively. Results: The study focused on the analysis of intracellular metabolites using NMR, LC-MS and GC-MS, with emphasis on the reproducibility and repeatability of the data. State of the art pre-processing and alignment tools and multivariate statistics were used to detect significantly altered levels of metabolites after exposing HepG2 cells to TCDD. Several metabolites identified using databases, literature and LC-nanomate-Orbitrap analysis were affected by the treatment. The observed changes in metabolite levels are discussed in relation to the reported effects of TCDD. Conclusions: Untargeted profiling of the polar and apolar metabolites of in vitro cultured HepG2 cells is a valid approach to studying the effects of TCDD on the cell metabolome. The approach described in this research demonstrates that highly reproducible experiments and correct normalization of the datasets are essential for obtaining reliable results. The effects of TCDD on HepG2 cells reported herein are in agreement with previous studies and serve to validate the procedures used in the present work. [ABSTRACT FROM AUTHOR]

Vegetables may protect against colorectal cancer (CRC) via changes in gene expression involved in anticarcinogenic mechanisms. There is considerable evidence that aberrant DNA methylation plays an important role in carcinogenesis. Furthermore, DNA methylation can be affected by dietary components. Therefore, in the present study, we investigated the DNA methylation status of CpG dinucleotides within the promoter region of the four genes protein kinase C β 1, ornithine decarboxylase 1, fos proto-oncogene and 5,10-methylenetetrahydrofolate reductase in the colon of female sporadic adenoma patients and healthy controls. These genes were chosen because their expression was modulated in response to altered vegetable intake, they are functionally relevant for CRC; they have CpG islands in their promoter region, and a methylation-specific restriction enzyme is available to permit quantitative assay. No significant differences in extent of methylation in colon DNA were detected for any of the four genes in both adenoma polyp patients and healthy controls after altering vegetable intake. Interestingly, before the intervention, ornithine decarboxylase 1 promoter methylation was lower in the colonic mucosa of the adenoma polyp patients when compared with healthy control subjects, which may explain the increased ornithine decarboxylase 1 activity in CRC reported in the literature. In conclusion, we found no evidence that changes in promoter methylation were responsible for differences in expression of four genes in the human colonic mucosa in response to altered vegetable intake. The mechanism(s) responsible for this altered gene expression and, indeed, potential effects on methylation of other genes remain to be determined. [ABSTRACT FROM PUBLISHER]

Worldwide, lung cancer is the most prevalent and lethal malignant disease. In addition to avoidance of the most predominant risk factor, i.e., tobacco use, consumption of high amounts of vegetables and fruits could be an effective means of preventing lung cancer. However, the molecular mechanisms underlying lung cancer risk reduction by vegetables are not clear. In the present study, the effect of vegetables on gene expression changes in the lungs of female C57Bl/6 mice was investigated using cDNA microarray technology. The mice were fed 1 of 8 diets for 2 wk: a control diet containing no vegetables (diet 1); a diet containing a vegetable mixture at 100 (diet 2, 10% dose), 200 (diet 3, 20% dose), or 400 (diet 4, 40% dose) g/kg; or a diet containing cauliflower at 70 (diet 5, 7% dose); carrots at 73 (diet 6, 7.3% dose); peas at 226 (diet 7, 22.6% dose); or onions at 31 (diet 8, 3.1% dose) g/kg. The vegetable mixture consisted of these 4 individual vegetables. After the mice were killed, the lungs were removed and total RNA was isolated from the lungs for expression analysis of 602 genes involved in pathways of (anti)-carcinogenesis. The results of this study suggest that individual vegetables have a higher potential of modulating genes (5 from the 8 modulated genes) in favor of lung cancer risk prevention, in comparison with the vegetable mixture (2 from the 7 modulated genes); the other gene modulations are expected to enhance lung cancer risk. The pathways involved were miscellaneous and included cell growth, apoptosis, biotransformation, and immune response. Furthermore, carrots were able to modulate most gene expressions, and most of these effects occurred in processes that favored lung cancer risk prevention. The current study provides more insight into the genetic mechanisms by which vegetables, in particular carrots, can prevent lung cancer risk. [ABSTRACT FROM AUTHOR]

There is abundant epidemiological evidence that vegetable consumption decreases colorectal cancer (CRC) risk. However, the molecular targets in the genome are mostly unknown. The present study investigated the effects of vegetable consumption on gene expression in the colon mucosa of female C57BI/6 mice using cDNA microarray technology. Mice were fed one of 8 diets: a control diet containing no vegetables (diet 1); a diet containing 100 g/kg (diet 2, 10% dose), 200 g/kg (diet 3, 20% dose), or 400 g/kg (diet 4, 40% dose) of a vegetable mixture; or a diet containing 70 g/kg of cauliflower (diet 5, 7% dose), 73 g/kg of carrots (diet 6, 7.3% dose), 226 g/kg of peas (diet 7, 22.6% dose); or 31 g/kg of onions (diet 8, 3.1 % dose). The vegetable mixture used in diets 2 to 4 consisted of the 4 individual vegetables used in diets 5 to 8: cauliflower (30% wet wt), carrots (30% wet wt), peas (30% wet wt), and onions (10% wet wt). To assess gene expression changes, colonic mucosal cells were collected after the mice were killed. Total ANA was isolated and microarray technology was used to measure the expression levels of 602 genes simultaneously. For 39 genes, significant dose-dependent effects were found, although in general the relations were not linear. For 15 genes, the altered expression could indeed explain reduced cancer risk at various stages of CRC development. Eleven genes were modulated by the vegetable mixture as well as by one or more of the individual vegetables. For 7 of the genes, the modulation by the mixture was due to the effect of a particular vegetable. These genes are of particular interest because they were consistently affected and could be involved in the prevention of CRC by vegetable consumption. [ABSTRACT FROM AUTHOR]

Occupational exposure to polycyclic aromatic hydrocarbons (PAH) increases the risk of developing lung cancer. Human exposure is often demonstrated by increased internal levels of PAH metabolites and of markers for early biological effects, like DNA adducts and cytogenetic aberrations. Objective: This study aimed to assess whether the current exposure to PAH of coke oven workers in a Dutch plant induced biological effects, and to determine if these effects are influenced by tobacco smoking and by genetic polymorphisms for the glutathione S-transferase genes GSTM1 and GSTT1. Methods: Urinary 1-hydroxypyrene (1-OHpyr) levels were used to monitor the internal dose, while the internal effective dose was assessed by monitoring PAH-DNA adducts, DNA strand breaks (Comet assay), sister-chromatid exchanges (SCE) and cells with a high frequency of SCE (HFC) in lymphocytes together with micronuclei (MN) in exfoliated urothelial cells. Results: Occupational exposure to PAH resulted in statistically significant increased 1- OHpyr levels (P 0.001), but it did not cause a significant induction of SCE, HFC, MN, DNA strand breaks or DNA adducts. Smoking caused a significant increase of 1-OHpyr (P 0.05), SCE (P 0.001), HFC (P 0.001) and DNA adducts (P 0.05), but not of MN or DNA strand breaks. Following correction for the smoking-related effects, no occupational induction of the effect biomarkers could be discerned. Multi-variate analysis did not show a significant influence of GSTM1 and GSTT1 polymorphisms on any biomarker. Also no significant interactions were observed between the various biomarkers. Conclusion: This study shows that in the examined plant, the occupational exposure to PAH does not result in measurable early biological effects [ABSTRACT FROM AUTHOR]

'Ribosome feedback' effects on the expression of the genes specifying tRNA and EF-Tu in E. coli have been studied at increased rrnB doses (rRNA gene doses). We confirm previous observations that the introduction into the cell of a multicopy plasmid carrying the rrnB operon reduces the cellular content of most tRNAs, including those encoded by the tRN A-tufB operon, but leaves the 5S rRNA content unaffected. Increase of the dosage of intact, but not of deleted rRNA genes, causes a slight drop in total EF-Tu that can be fully accounted for by a decrease in EF-TuB level. The drop in EF-TuB content (approx, 25%) is much smaller than that in tRNA content (approx. 80%). The synthesis rate of total EF-Tu is hardly affected, indicating that the turnover of EF-Tu has not changed. The ratio of tRNA over tuf RNA synthesis rates remains the same after elevation of rrnB dosage, Considering the large decrease in tRNA content this means that both RNA synthesis rates decrease to approximately the same extent. The relatively small drop in EF-Tu synthesis must be due, therefore, to an enhancement of the number of EF-Tu molecules synthesized per mRNA molecule. Apparently a post-transcriptional mechanism, regulating EF-Tu synthesis, becomes operative under these conditions. Growth-rate-dependent regulation of the tRNA-tufB/ operon has been studied using lysogens carrying tRNA′:lacZ and tRNA-tufB:lacZ operon fusions and a tufB′:lacZ′ gene fusion. These experiments show that the cellular contents of tRNA, tufB RNA and EF-TuB vary in direct proportion to the growth rate. This indicates that growth rate control of tRNA-tufB operon transedption resembles that of stable RNA operons and not of r-protein operons, and that the read-through of the terminator at the end of the tRNA gene eluster remains unaltered. [ABSTRACT FROM AUTHOR]

The mechanism underlying feedback inhibition of tufB expression has been studied in vivo by gene-dosage experiments and by gene and operon fusions involving lacZ. Raising the cellular EF-Tu content, by introducing a multicopy plasmid encoding EF-TuA into the cell, repressed the level of EF-TuB but left the content of tRNAThr3, encoded by the tRNA-tufB operon, unaffected. This indicates that autoregulation of chromosomal tufB expression does not occur by modulating transcription initiation at the promoter of the tRNA-tufB operon. This conclusion is further substantiated by experiments with a tRNA′:lacZ operon fusion. The molecular ratio of chromosome-borne tufA and tufB transcripts also remained unaltered under conditions of excess EF-Tu, though experiments with a tRNA-tufB′:lacZ operon fusion showed a decrease of tufB transcripts. Our data further exclude drastic effects of the autogenous repressor on processing of the contranscript of the operon into monocistronic tufB RNA and on alteration of EF-TuB turnover. Two possible mechanisms remain, which cannot yet be decided between. One is modulation of EF-Tu by transcription termination either directly or indirectly by affecting antitermination. The second is translational repression. In vitro translation of transcripts derived from SP6 clones did not reveal any feedback inhibition of EF-TuB synthesis. Surprisingly, addition of EF-Tu to a coupled transcription/translation systems was found to block transcription initiation at the primary promoter of the tRNA-tufB operon by over 90%. Although this in vitro effect of EF-Tu could not be demonstrated in vivo, possibly because of a difference in higher-order structure between plasmid-borne and chromosome-borne DNA, it indicates that under certain conditions EF-Tu binds very specifically to the tRNA-tufB operon promoter or its upstream region. [ABSTRACT FROM AUTHOR]

The expression of tufB, one of the two EF-Tu-encoding genes in Escherichia coli, is under autogenous control. Feedback inhibition of tufB expression by plasmid-borne EF-Tu has been used to answer the question of whether or not the integrity of the guanine-nueleotide-binding domain of EF-Tu is required for the autoregulatory role of the factor protein. We show that a large deletion of tufB, causing the elimination of an 81-amino-acid segment from the plasmid-borne EF-Tu, does not abolish tufB repression. We conclude that the autoregulation of the cellular EF-Tu level is not dependent on an intact guanine-nueleotide-binding domain and does not require binding of GTP to EF-Tu. The repressor activity of the deletion derivative of EF-Tu can be measured despite a rapid disappearance of the (altered) mutant protein from the soluble cytoplasmic fraction of the cell. Degradation and assembly in larger complexes are responsible for this disappearance. [ABSTRACT FROM AUTHOR]

An investigation is presented of occupational exposure to polycyclic aromatic hydrocarbons (PAH) in a carbon-electrode manufacturing plant, as assessed by three monitoring methods, viz. environmental monitoring of the external dose by analysis of personal air samples, biological monitoring of the internal dose by analysis of urinary 1-hydroxypyrene (1-OHpyrene), and biological effect monitoring by dosimetry of PAH-DNA adducts in blood lymphocytes. On the basis of job conditions, workers at the plant were divided into three groups with presumed low, intermediate and high exposure to air-borne PAH, respectively. All air samples showed levels of total PAH below the current MAC-value in the Netherlands, which is 200µg/m3, whereas the benzo[a]pyrene level was occasionally higher than the recommended concentration of 2 µg/m3 The values of 1-OHpyrene in urine from the intermediate and high exposure groups were significantly higher than those of the low exposure group, namely 3.6- and 8.2-fold, respectively. Clear external and internal exposure was thus demonstrated for workers of the high and intermediate exposure groups, but this did not result in a measurable effect at the DNA level in blood lymphocytes. Tobacco smoking, on the other hand, caused a significant increase of the levels of PAH-DNA adducts but did not affect 1 -OHpyrene values. These data suggest that smoking is a more important risk factor for adverse health effects, i.e. cancer, than occupational exposure to PAH in this plant. © 1998 British Occupational Hygiene Society. Published by Elsevier Science Ltd. [ABSTRACT FROM AUTHOR]