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Monuron (1,1-dimethyl-3-(4-chlorophenyl)urea) is a non-selective phenylurea herbicide, widely used in developing countries although concerns have been raised about its toxicity and carcinogenicity. Monuron was evaluated by the National Toxicology Program in 1988 and shown to be a male rat-specific renal carcinogen. We report that oral administration of Monuron to male rats for 3 days, leads to a larger number of genes being differentially expressed in the renal-cortex than in the liver. Further, we observed up-regulation of cell cycle genes and genes involved in cell proliferation in the renal-cortex while in the liver xenobiotic metabolising enzymes were up-regulated. We also identified one commonly down-regulated gene in both organs – fragile histidine triad gene (Fhit), a putative tumour suppressor gene; however the down-regulation was only significant at the protein level in the liver. In addition, we conducted in vitrowhole-genome transcriptomics studies with human and rat renal cortical cells. Rat cells exposed to Monuron showed down-regulation of sterol biosynthesis, spliceosome and cell cycle genes and up-regulation of genes involved in amino acid metabolism and transport. No genes were found to be differentially expressed in human cells exposed to Monuron. Overall, the findings from the in vitrostudies showed very little overlap with the whole animal findings.

In this study, we developed a transcriptomics based human in vitro model for predicting DILI in humans. The transcriptomics data (Affymetrix GeneChip Human Genome U133 Plus 2.0) from primary human hepatocytes were provided by the Japanese Toxicogenomics Project (TGP). The selected compounds were divided into two groups, i.e., most-DILI and no-DILI, based on FDA-approved drug labels. The compounds were further grouped in a training and validation set. The training set, containing the most extreme most-DILI and no-DILI compounds based on the in vivo rat clinical chemistry measurements from TGP, was used to develop the prediction model. The validation set showed high accuracy (> 90%) and performed better than splitting the compounds into training and validation set randomly.

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 KBrO3resulted 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 KBrO3exposure, 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 KBrO3cause 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 KBrO3exposure.

There is a need to develop quick, cheap, sensitive and specific methods to detect the carcinogenic potential of chemicals. Currently there is no in vitromodel system for reliable detection of non-genotoxic carcinogens (NGTX) and current tests for detection of genotoxic carcinogens (GTX) can have low specificity. A transcriptomics approach holds promise and a few studies have utilised this technique. However, the majority of these studies have examined liver carcinogens with little work on renal carcinogens which may act viarenal-specific NGTX mechanisms. In this study the normal rat renal cell line (NRK-52E) was exposed to sub-toxic concentrations of selected rat renal carcinogens and non-carcinogens (NC) for 6 h, 24 h and 72 h. Renal carcinogens were classified based on their presumed mode of action into GTX and NGTX classes. A whole-genome transcriptomics approach was used to determined genes and pathways as potential signatures for GTX, NGTX and those common to both carcinogenic events in vitro. For some of the GTX compounds an S9 drug metabolising system was included to aid pro-carcinogen activation. Only three genes were commonly deregulated after carcinogen (GTX + NGTX) exposure, one Mdm2with a detection rate of 67%, and p21and Cd55with a detection rate of 56%. However, examination of enriched pathways showed that 3 out of 4 NGTX carcinogens and 4 out of 5 GTX carcinogens were related to known pathways involved in carcinogenesis giving a detection rate of 78%. In contrast, none of the NC chemicals induced any of the above genes or well-established carcinogenic pathways. Additionally, five genes (Lingo1, Hmox1, Ssu72, Lyrmand Usp9x) were commonly altered with 3 out of 4 NGTX carcinogens but not with NC or GTX carcinogens. However, there was no clear separation of GTX and NGTX carcinogens using pathway analysis with several pathways being common to both classes. The findings presented here indicate that the NRK-52E cell line has the potential to detect carcinogenic chemicals, although a much larger number of chemicals need to be used to confirm these findings.

Paternal exposure to genotoxic compounds is thought to contribute to diseases in their offspring. Therefore, it is of importance to develop biomarkers of male germ cell exposure to genotoxins. Unfortunately, the testis cannot be reached for routine biomonitoring, but mRNA-profiles in spermatozoa may reflect the processes that have occurred in the testis after exposures to genotoxins, since spermatozoa are largely transcriptionally inactive. Therefore, mRNA profiles from sperm in ejaculates of cigarette smokers (N=4) were compared with non-smokers (N=4). Smoking behaviour was verified by assessing cotinine levels in seminal plasma. High expression of the germ cell specific gene protamine 2 (PRM2) was observed in spermatozoal mRNA isolates by Q-PCR, which was absent in reference mRNA isolates obtained from a pool of other organs. Gene-expression analysis was subsequently performed using microarray technology and a total of 781 genes were found to be differentially expressed in spermatozoa of smokers compared to non-smokers (fold change >40%; p<0.05). To further limit the number of false positive results, genes were additionally selected on basis of the correlation between their expression levels with cotinine concentrations in seminal plasma (r>0.80 as arbitrary cut-off value, p<0.05), and a total of 200 transcripts remained, of which the germ cell specific transcription factor SALF was the highest up-regulated gene (5.4-fold) and the zinc finger encoding gene TRIM26 most down regulated (7.4-fold). Although no altered pathways could be identified for the differentially expressed genes, an enrichment was observed for NF-κB regulated genes (46% vs. 27%, p=0.004) playing a central role in stress response. Accordingly, subsequent analysis of transcription factor networks suggests that apoptosis was inhibited in smokers. These data show the feasibility of using gene-expression profiles in mature sperm to elucidate gene–environment interactions in male testis.

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.

Sandwich-cultured primary rat hepatocytes are often used as an in vitro model in toxicology and pharmacology. However, loss of liver-specific functions, in particular, the decline of cytochrome P450 (P450) enzyme activity, limits the value of this model for prediction of in vivo toxicity. In this study, we investigated whether a hepatic in vitro system with improved metabolic competence enhances the predictability for coumarin-induced in vivo toxicity by using a toxicogenomics approach. Therefore, primary rat hepatocytes were cultured in sandwich configuration in medium containing a mixture of low concentrations of P450 inducers, phenobarbital, dexamethasone, and {szligbeta}-naphthoflavone. The toxicogenomics approach used enabled comparison of similar mechanistic end-points at the molecular level between in vitro and in vivo conditions, namely, compound-induced changes in multiple genes and signaling pathways. Toxicant-induced cytotoxic effects and gene expression profiles observed in hepatocytes cultured in modified medium and hepatocytes cultured in standard medium (without inducers) were compared with results from a rat in vivo study. Coumarin was used as a model compound because its toxicity depends on bioactivation by P450 enzymes. Metabolism of coumarin toward active metabolites, coumarin-induced cytotoxicity, and gene expression modulation were more pronounced in hepatocytes cultured in modified medium compared with hepatocytes cultured in standard medium. In addition, more genes and biological pathways were similarly affected by coumarin in hepatocytes cultured in modified medium and in vivo. In conclusion, these experiments showed that for coumarin-induced toxicity, sandwich-cultured hepatocytes maintained in modified medium better represent the situation in vivo compared with hepatocytes cultured in standard medium.

Polycyclic aromatic hydrocarbons (PAHs) can occur in relatively high concentrations in the air, and many PAHs are known or suspected carcinogens. In order to better understand differences in carcinogenic potency between PAHs, we investigated modulation of gene expression in human HepG2 cells after 6 h incubation with varying doses of benzo[a]pyrene (B[a]P), benzo[b]fluoranthene (B[b]F), fluoranthene (FA), dibenzo[a,h]anthracene (DB[a,h]A), 1-methylphenanthrene (1-MPA) or dibenzo[a,l]pyrene (DB[a,l]P), by using cDNA microarrays containing 600 toxicologically relevant genes. Furthermore, DNA adduct levels induced by the compounds were assessed with 32P-post-labeling, and carcinogenic potency was determined by literature study. All tested PAHs, except 1-MPA, induced gene expression changes in HepG2 cells, although generally no dose–response relationship could be detected. Clustering and principal component analysis showed that gene expression changes were compound specific, since for each compound all concentrations grouped together. Furthermore, it showed that the six PAHs can be divided into three groups, first FA and 1-MPA, second B[a]P, B[b]F and DB[a,h]A, and third DB[a,l]P. This grouping corresponds with the carcinogenic potencies of the individual compounds. Many of the modulated genes are involved in biological pathways like apoptosis, cholesterol biosynthesis and fatty acid synthesis. The order of DNA adduct levels induced by the PAHs was: B[a]P ≫ DB[a,l]P > B[b]F > DB[a,h]A > 1-MPA ≥ FA. When comparing the expression change of individual genes with DNA adduct levels, carcinogenic potency or Ah-receptor antagonicity (the last two were taken from literature), several highly correlated genes were found, of which CYP1A1, PRKCA, SLC22A3, NFKB1A, CYP1A2 and CYP2D6 correlated with all parameters. Our data indicate that discrimination of high and low carcinogenic PAHs by gene expression profiling is feasible. Also, the carcinogenic PAHs induce several pathways that were not affected by the least carcinogenic PAHs.

The evidence from epidemiological and experimental studies that vegetables reduce the risk of colorectal cancer is convincing. However, the involved genes and genetic pathways are not clear. The aim of this study was to identify genes that are modulated in vivo in colorectal mucosa by vegetables, and to investigate whether colon adenoma patients respond differently compared with healthy controls. Twenty female adenoma patients and eight healthy controls were randomly split into two groups of ten and four persons, respectively, receiving either a 50% decreased (=75 g/day) or doubled (=300 g/day) intake of vegetables for 2 weeks. In order to assess the effects on gene expression at the target level, colorectal biopsies were collected before and after the intervention. Total RNA was isolated from the biopsies to measure gene expression of 597 genes relevant for responses to xenobiotics by microarray technology, followed by confidence analyses to identify differentially expressed genes. Mainly genes related to cell cycle control and genes for oxidoreductase activities were over-represented in the list of modulated genes. Twenty genes were modulated, which are known to be related to (colon)carcinogenesis. Seven genes were similarly modulated in patients and controls, for example fos proto-oncogene and ornithine decarboxylase. Thirteen genes were modulated differently in patients compared with controls, including cyclooxygenase-2 and human mdm2-A in patients and cytochrome P45027B1, -2C19, -2D6, -2C9 and -3A4 in controls. Almost all the effects on modulating the expression of genes by altering vegetable intake can be mechanistically linked to cellular processes that explain either prevention of colorectal cancer risk by high vegetable intake or increased colorectal cancer risk by low vegetable intake. Furthermore, it seems that vegetables in patients affect genes involved in the late stage of colorectal cancer, whereas in controls genes involved in the initiation phase are modulated.

Methyl-DNA adducts are induced by a number of lifestyle, environmental and occupational carcinogens, however knowledge about their kinetics is scarce. Here, N7-methylguanine (N7-MeGua) and O6-methylguanine (O6-MeGua) levels were determined in the DNA of white blood cells from eight cancer patients treated hr with the antitumour drug dacarbazine (DTIC). Five of the patients were treated with the drug as a single agent (a single dose of 800 mg m-2) and three on three successive days with dacarbazine (225 mg m-2 day-1) in combination with other drugs. The data indicate that maximum adduct levels are reached at 4-8 h after treatment and that the amount of N7-MeGua is at least 20-fold higher than that of O6-MeGua. The half-life of N7-MeGua is 40-96 h and that of O6-MeGua 25-27 h. Following treatment on three consecutive days, an accumulation of N7-MeGua was observed but not of O6-MeGua. The data show substantial interindividual differences in adduct levels but not in the ratio of N7/O6-MeGua. This may reflect differences in the metabolism of dacarbazine or in repair capacities.

Comparison of results derived from transgenic animal gene-mutation assays with those from mutation analyses in endogenous genes is an important step in the validation of the former. We have used λlacZtransgenic mice to study alkylation-induced mutagenesis in vivo in (a) lacZand hprtin spleen cells, and (b) lacZand Dlb-1in small intestine from λlacZ+/0/Dlb-1a/bmice. Induction of mutations by ethyl- and methylnitrosourea (ENU, MNU) and ethyl methanesulphonate (EMS) was investigated at 7 weeks after a single i.p. dose of each of these chemicals. In the small intestine, treatment with various dosages of ENU (10–150 mg/kg) resulted in a linear dose-response in both lacZand Dlb-1. MNU (30 mg/kg) was also mutagenic in lacZand Dlb-1, while EMS (250 mg/kg) did not significantly induce mutations in either gene. In spleen, ENU gave a linear dose-related response in both lacZand hprt, MNU induced mutations in both lacZand hprt, and EMS was only positive for lacZ. No differences in response were observed between single and split-dose treatment with ENU (1×50 or 5×10 mg/kg with a 1- or 7-day interval), both in spleen and small intestine, except for lacZin small intestine, where the single high dose gave a significantly higher induction than the split dose with the 7-day interval. The overall results suggest that mutagenic effects of fractionated doses are generally additive. In most cases, the induction factor (ratio treated over controls) for mutations in lacZwas lower than that for hprtand Dlb-1, presumably due to a higher background in lacZand/or a lower mutability of lacZ. The general concordance between the data for lacZand the endogenous genes indicates that λlacZtransgenic mice are a suitable model to study induction of gene mutations in vivo.

The recent introduction of the phenyl-β-d-galactopyranoside (P-gal)-based positive-selection system for screening of λlacZ phages originating from the λlacZ transgenic mouse (Muta Mouse) has made the determination of mutant frequencies (MF) a much simpler task. Previously, MF data from these mice have been collected by means of the 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) colour-screening procedure. To determine whether data obtained with the two systems are comparable, the MF in λ phages recovered from liver and brain of transgenic mice treated with N-ethyl-N-nitrosourea (ENU) and liver of benzo(a)pyrene (B(a)P)-treated mice was determined with both procedures. For the livers of mice treated with ENU, both methods yielded approximately the same MF values. No induction of mutants, relative to the control animals, was seen after 1.5 h, but a clear 4-fold increase was measured with both assays at the 14-day time point. No induction of mutants was found in the brain with either method. In the B(a)P-treated mice, both methods showed a substantial induction in MF after 21, 28 and 35 days. The values generated by the X-gal and P-gal methods were not significantly different, with the exception of the 35-day post-treatment point that appeared higher in the X-gal assay. When the mutants isolated by use of the X-gal method were tested in the P-gal assay, a number of these did not turn up as mutants, and the significance disappeared. In conclusion, the data obtained with the two screening procedures agree to such an extent as to permit a direct comparison between the earlier results generated with X-gal and P-gal values generated with the new positive-selection method. This is likely to apply also to other organs and mutagens than those studied here.

To investigate tissue‐specific relations between DNA adducts and mutagenesis in vivo, λlacZ transgenic mice were treated i.p. with N‐ethyl‐N‐nitrosourea (ENU), diethylnitrosamine (DEN), and ethyl methanesulphonate (EMS). In liver, bone marrow, and brain DNA from mice sacrificed at several time points after treatment O6‐ethylguanine (O6‐EtG) and N7‐ethylguanine (N7‐EtG) levels were determined as well as the mutant frequency (MF) in lacZ. In liver DNA of ENU‐ and DEN‐treated mice, the bulk of O6‐EtG was removed at 3 days after treatment, while the MF continued to increase thereafter. This suggests that O6‐EtG is not the major premutagenic lesion in the liver. Indeed, sequence analysis of mutants showed only 24% GC → AT transitions, consistent with the O6‐EtG lesion, and 28% TA → AT transversions, expected from O2‐ethylthymine. In bone marrow after ENU treatment, a maximum mutation induction occurred at 3 days post‐treatment, of which 43% were GC → AT mutations and 22% were TA → AT mutations. This suggests that in bone marrow O6‐EtG may be a major premutagenic lesion at the 3‐day time point. In liver and bone marrow, EMS treatment gave rise to a high level of N7‐EtG and a low level of O6‐EtG but no increase in MF. No adducts or mutation induction were observed in bone marrow of DEN‐treated mice. No MF increase was observed in the brain of either ENU‐ or EMS‐treated mice, although O6‐ and N7‐adducts were present. Environ. Mol. Mutagen. 31:18–31, 1998. © 1998 Wiley‐Liss, Inc.

The determination of thelacZ mutant frequency in λgt10lacZ phage vectors isolated from the transgenic mouse strain 40.6 (MutaMouse), requires the screening of large numbers of phages on β-galactosidase activity. Existing methods rely on distinguishing a few white plaques on X-gal containing plates amongst a multide of blue ones which is both time-consuming and expensive. The new screening method described here employs the galactose sensitiveEscherichia coli C lacZ recA galE strain into which a multicopy plasmid has been introduced, which results in over-expression of thegalK andgalT genes. In the presence of phenyl-β-d-galactopyranoside, a substrate for β-galactosidase, this leads to the suppression of λlacZ+ phage propagation without affecting the ability of λlacZ− phages to form plaques. With this method it is possible to screen 1.5×106 phages on a single 9-cm Petri dish. Furthermore, the need for blue/white screening has been eliminated.