Unique patterns of gene expression changes in liver after treatment of mice for 2 weeks with different known carcinogens and non-carcinogens
Mari Iida1,5,
Colleen H. Anna1,
Wanda M. Holliday1,
Jennifer B. Collins2,
Michael L. Cunningham3,
Robert C. Sills4 and
Theodora R. Devereux1,6
1 Laboratory of Molecular Carcinogenesis, 2 National Center for Toxicogenomics, 3 Laboratory of Pharmacology and Chemistry and 4 Laboratory of Experimental Pathology, National Institute of Environmental Health Sciences, National Institute of Health, Research Triangle Park, NC 27709, USA
6 To whom correspondence should be addressed at: Laboratory of Molecular Carcinogenesis, Mail Drop D4-04, National Institute of Environmental Health Sciences, PO Box 12233, Research Triangle Park, NC 27709, USA. Tel: +1 919 541 3421; Fax: +1 919 541 0146; Email: devereux{at}niehs.nih.gov
 |
Abstract
|
---|
Previously we demonstrated that the mouse liver tumor response to the non-genotoxic carcinogens oxazepam and Wyeth-14,643 involved more differences than similarities in changes in early gene expression. In this study we used quantitative real-time PCR and oligonucleotide microarray analysis to identify genes that were up- or down-regulated in mouse liver early after treatment with different known carcinogens, including oxazepam (125 and 2500 p.p.m.), o-nitrotoluene (1250 and 5000 p.p.m.) and methyleugenol (75 mg/kg/day), or the non-carcinogens p-nitrotoluene (5000 p.p.m.), eugenol (75 mg/kg/day) and acetaminophen (6000 p.p.m.). Starting at 6 weeks of age, mice were treated with the different compounds for 2 weeks in the diet, at which time the livers were collected. First, expression of 12 genes found previously to be altered in liver after 2 weeks treatment with oxazepam and/or Wyeth-14,643 was examined in livers from the various chemical treatment groups. These gene expression changes were confirmed for the livers from the oxazepam-treated mice in the present study, but were not good early markers for all the carcinogens in this study. In addition, expression of 20 842 genes was assessed by oligonucleotide microarray [n = 4 livers/group, 2 hybridizations/liver (with fluor reversals)] and the results were analyzed using the Rosetta Resolver System and GeneSpring software. The analyses revealed that several cancer-related genes, including Fhit, Wwox, Tsc-22 and Gadd45b, were induced or repressed in unique patterns for specific carcinogens and not altered by the non-carcinogens. The data indicate that even if the tumor response, including molecular alterations, is similar, such as for oxazepam and methyleugenol, early gene expression changes appear to be carcinogen specific and seem to involve apoptosis and cell cycle-related genes.
Abbreviations: QRT-PCR, quantitative real-time PCR
 |
Introduction
|
---|
The 2 year rodent bioassay is one of several evaluations that are widely used by governmental regulatory agencies as well as pharmaceutical and chemical companies to determine the toxic and carcinogenic potential of chemicals or environmental agents where human exposure is anticipated. Two year rodent studies have identified potential human carcinogens (1) and, in some cases, these have been confirmed with epidemiological evidence. Those studies are long-term, expensive undertakings that utilize primarily the tumor response data to estimate risk. The use of global gene expression studies provides an opportunity to understand the early steps in carcinogenesis, to identify early biomarkers of carcinogenesis and to distinguish carcinogens from non-carcinogens in a faster, more cost-effective manner.
B6C3F1 mice are one of the rodent models commonly used in 2 year bioassays (1). Both genotoxic and non-genotoxic carcinogens cause cancer in the mouse liver more frequently than any other site for both rats and mice. We and other investigators have identified alterations in cancer-related genes or proteins in liver tissues/tumors induced by certain carcinogens (27) and our recent studies have concentrated on understanding the mechanisms of non-genotoxic or weakly genotoxic carcinogens. While some processes, such as blocking of apoptosis and accelerated cell cycle progression, have been implicated in non-genotoxic hepatocarcinogenesis (8,9), the mechanisms of the early stages of hepatocarcinogenesis have not been fully defined and few studies have compared changes across multiple carcinogens. The identification of differentially regulated genes in liver following treatment with different chemicals at early time points may prove critical for understanding chemically induced hepatocarcinogenesis, especially for non-genotoxic carcinogens.
We have reported changes in global gene expression in livers from B6C3F1 mice following treatment with a carcinogenic dose of the anti-anxiety drug oxazepam (2500 p.p.m.) or the peroxisome proliferator Wyeth-14,643 (500 p.p.m.) for 2 weeks or 6 months compared with age-matched untreated mice (7). Expression of 25 and 36 genes were changed relative to control livers by oxazepam treatment and 126 genes and 220 genes were altered by Wyeth-14,643 treatment at 2 weeks and 6 months, respectively. Much of this difference appeared to be due to the effect of Wyeth-14,643 as a potent peroxisome proliferator and activator of many enzymes involved in ß-oxidation of fatty acids and lipid metabolism. The data from that study also showed that many of the changes in expression associated with processes involved in carcinogenesis identified at 2 weeks after treatment continued until 6 months and were also found in the liver tumors (7).
In the present study we utilized tumor response data from the National Toxicology Program (NTP) to identify sets of structurally related carcinogens and non-carcinogens for analysis. For example, o-nitrotoluene and methyleugenol are carcinogens, while p-nitrotoluene and eugenol were not found to be carcinogenic in 2 year rodent studies (10,11).
Oxazepam is commonly used as an anti-anxiety drug and is a non-genotoxic mouse liver carcinogen (2,6,7,10). All male and female mice in the high dose (2500 p.p.m.) groups in the NTP study developed liver tumors (11). Even though the tumor response in the low dose (125 p.p.m.) groups was not statistically different from the controls, the H-ras mutation profile in these tumors was significantly different from spontaneous tumors, suggesting that there was a weak carcinogenic response even at that low dose (10). Oxazepam was chosen because it was utilized in our previous study but with a different study design. In addition, we utilized both sexes and two doses of oxazepam and o-nitrotoluene to try to identify changes potentially associated with the carcinogenic process.
Nitrotoluenes, including o-nitrotoluene and p-nitrotoluene, are high production chemicals in the USA and are used to synthesize many industrial products (12,13). The incidence of hepatocellular adenomas/carcinomas after treatment with 5000 p.p.m. o-nitrotoluene was increased in female mice, but not in male mice. Exposure of both male and female mice to 5000 p.p.m. o-nitrotoluene caused the development of massive hemangiosarcomas, and this led to low survival of the male mice and a low incidence of hepatocellular tumor formation. Treatment of B6C3F1 mice with p-nitrotoluene did not result in liver tumor formation in the 2 year bioassay.
Methyleugenol is found in a variety of food products and essential oils, is structurally similar to the known carcinogen safrole and is a hepatocarcinogen in both male and female mice (14), while the evidence for hepatocarcinogenicity of eugenol was equivocal. Both methyleugenol and safrole lack freely available functional groups for conjugation reactions, while eugenol can undergo conjugation reactions directly because it contains free hydroxyl groups, and thus is likely to be detoxified more efficiently (15).
Acetaminophen is widely used as an analgesic and antipyretic drug throughout the world. Even though acetaminophen is non-carcinogenic, it is toxic at higher doses and can lead to liver and kidney failure and, in severe cases, death. Based on NTP studies (16) acetaminophen was administered to mice as another non-carcinogenic control group.
The purposes of this study were: (i) to gain a better understanding of mechanisms of chemically induced mouse liver carcinogenesis; (ii) to find biomarkers and early expression changes that may help predict hepatocarcinogenic activity in liver; (iii) to determine whether changes in expression of small sets of early genes can distinguish classes of carcinogens. For cost and time considerations both sexes were not included for all treatments. For example, p-nitrotoluene, eugenol and acetaminophen are all known to be non-carcinogens and we chose to study only females in this study for these treatments. On the other hand, because of our former studies on the molecular carcinogenesis of oxazepam (6,8), comparisons of gene expression in livers from males and females after treatment with oxazepam may help us focus on potentially important early cancer-associated changes.
 |
Materials and methods
|
---|
Animals
Four 6-week-old male or female B6C3F1 mice for each treatment and untreated control group were obtained from Taconic (Germantown, NY) and kept on a 12 h light/dark cycle (lights on 6 a.m.6 p.m.). The chemicals and doses were chosen based on prior NTP studies because the carcinogens were either non-genotoxic or weakly genotoxic and induced a strong hepatocellular tumor response in the B6C3F1 mouse. We also wanted to compare structurally similar non-carcinogens. Chemical dose information and genotoxicity and carcinogenicity data are shown in Table I. The treatment groups included: both male and female mice treated for 2 weeks with oxazepam at doses of 125 and 2500 p.p.m.; female mice dosed with 1250 or 5000 p.p.m. o-nitrotoluene or 5000 p.p.m. p-nitrotoluene; male mice dosed with 5000 p.p.m. o-nitrotoluene; female mice dosed with methyleugenol or eugenol at 75 mg/kg/day; female mice dosed with 6000 p.p.m. acetaminophen. Beginning on a Wednesday, all animals were dosed in the feed except for the methyleugenol and eugenol groups, which were dosed by gavage daily at 910 a.m. Monday to Friday. All mice were killed between 9 and 10 a.m. 2 weeks after the start of the treatment. Age-matched untreated male and female mice were used as controls. All animal procedures were performed under the highest standards of humane care in accordance with protocols approved by the NIEHS committee on animal care and the NIH Guide for the Care and Use of Laboratory Animals.
At necropsy the mice were weighed, killed with CO2 from a regulated source and the left lateral lobe of each liver was harvested, weighed, chopped into small pieces, flash-frozen in liquid N2 and stored at 80°C until subsequent RNA isolation or protein extraction. One other section of each left lobe of liver was fixed in 10% neutral buffered formalin, embedded in paraffin and cut into 5 µm sections for histological staining. One specimen was stained with hematoxylin and eosin for routine histological examination.
RNA isolation, direct label protocol and feature extraction
Total liver RNA was isolated with a RNeasy Midi Kit (Qiagen, Valencia, CA) according to the manufacturer's protocols. The quality of the RNA was evaluated by measuring the 260:280 nm absorbance ratio, utilizing formaldehyde agarose gel electrophoresis and an Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA). Gene expression analysis was conducted using the Agilent Mouse Oligo array of
20 000 genes, which contains toxicology markers from NIEHS, the Toxicogenomics Research Consortium and Paradigm Genetics.
Ten micrograms of RNA from each sample was labeled using the Agilent Fluorescent Direct Label Kit protocol. Hybridizations were performed for 16 h in a rotating hybridization oven using the Agilent 60mer oligo microarray processing protocol. Slides were washed as indicated in this protocol and then scanned with an Agilent Scanner.
Two hybridizations with fluor reversals were performed for each liver RNA sample and four individual livers were used from each chemical treatment group. The control samples were pools of RNA from 10 individual livers and the RNA was isolated from each individual liver and tested for quality prior to mixing. Females in treated groups were compared with female controls and males in treated groups with male controls.
Microarray expression data were obtained using the Agilent Feature Extraction software, using defaults for all parameters except the Ratio terms. To account for the use of the Direct Label protocol, the error terms were changed, as suggested by Agilent Technology, to the following: Cy5 multiplicative error = 0.15; Cy3 multiplicative error = 0.25; Cy5 additive error = 20; Cy3 additive error = 20.
Rosetta Resolver (v3.2.2)
Images and GEML files, including error and P values, were exported from the Agilent Feature Extraction software and deposited into Rosetta Resolver (version 3.2, build 3.2.2.0.33) (Rosetta Biosoftware, Kirkland, WA). The resultant ratio profiles were combined into ratio experiments as described in Stoughton and Dai (2002). Intensity plots were generated for each ratio experiment and genes were considered signature genes if P < 0.05.
Microarray analysis programs
Expression analysis for all replicate microarray experiments was performed with GeneSpring 6.2 (Silicon Genetics, Redwood City, CA). Comparisons of gene expression across treatment groups were performed using Venn Diagrams and clustering was performed with the Condition Tree algorithm. In addition, the Gene Ontology groupings and Gen Mapps 2.0 program were used in conjunction with Gene Spring to identify pathways and functional groups of genes. The Ingenuity program (Ingenuity Systems, Mountain View, CA) was also utilized to identify networks of interacting genes and other functional groups.
Reverse transcription
To remove genomic DNA, RNA samples were incubated with 1 U/µg RNA of RNase-free DNase I (Invitrogen Corp.) for 15 min at room temperature. The DNase I was then inactivated by addition of 2.5 mM EDTA (pH 8.0) and heating at 65°C for 10 min. Reverse transcription of RNA (1 µg) using MuLV was carried out according to the manufacturer's instructions using oligodeoxythymidine primers (Applied Biosystems, Foster City, CA). As a negative control, a sample containing RNA but no reverse transcriptase (RT) was also included. A 1:10 dilution of this cDNA with DNase-free water was used for real-time PCR analysis in 96-well plates.
Real-time PCR analysis
Quantitative gene expression levels were determined using real-time PCR with the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA) and SYBR Green I dye or TaqMan MGB probes (FAMTM dye labeled). Primers (7) were created using Applied Biosystems Primer Express Software version 2.0 or purchased by Applied Biosystems Assays-on-Demand Gene expression products. For amplification diluted cDNA was combined with a reaction mixture containing SYBR Green PCR core reagents (Applied Biosystems, catalog no. 4304886) or with TaqMan universal PCR Master Mix (Applied Biosystems, catalog no. 4304437) according to the manufacturer's instructions. Samples were analyzed in duplicate and a sample without reverse transcriptase was included with each plate to detect contamination by genomic DNA. Amplification was carried out as follows: 50°C for 2 min (for uracil N-glycosylase incubation); 95°C for 10 min (denaturation); 40 cylces of 95°C for 15 s and 60°C for 30 s (denaturation/amplification). For SYBR Green I dye reactions dissociation curves were also created by adding the following steps to the end of the amplification reaction: 95°C for 15 s (denaturation) and 60°C for 20 s, then gradually increasing to 95°C over 20 min, with a final hold at 95°C for 15 s. Fold increases or decreases in gene expression were determined by quantitation of cDNA from target (treated) samples relative to a calibrator sample (untreated). The ß-actin gene was used as the endogenous control for normalization of initial RNA levels. To determine this normalized value, 2
CT values were compared between target and calibrator samples, where the change in crossing threshold (
Ct) = Cttarget gene Ctß-actin and 
Ct =
Ctcontrol
Cttreatment.
Western blot analysis
Cellular proteins were extracted from frozen tissues in radioimmunoprecipitation assay buffer as described previously (7). Equal amounts of total protein (50 µg) were denatured by boiling in Laemmli sample buffer and were electrophoresed (200 V, room temperature) on 15% acrylamide gels (Bio-Rad, Hercules, CA) using Mini Protean II electrophoresis tanks (Bio-Rad). After electrophoresis, proteins were transferred (240 mA for 1 h at room temperature) to 0.45 µm Immobilon-P PVDF membranes (Millipore, Bedford, MA). The PVDF membranes were blocked for 1 h with 5% non-fat dry milk in phosphate-buffered saline. After blocking the membranes were incubated with each primary antibody overnight as follows: mouse monoclonal Fhit antibody (clone 30, catalog no. 611740; BD Biosciences, San Diego, CA), mouse monoclonal EGF receptor antibody (clone 13, catalog no. 610016; BD Biosciences) and anti-Wwox antibody (a gift from Drs Akira Watanabe and Hiroyuki Aburatani, University of Tokyo, Japan). Membranes were washed with Tris-buffered saline containing Tween 20 (10 mM TrisHCl, pH 7.5, 150 mM NaCl and 0.01% Tween 20) and exposed to the appropriate secondary antibody (1:5000 for horseradish peroxidase-conjugated anti-mouse IgG; Santa Cruz, Santa Cruz, CA). Specific proteins were visualized using the ECL system (Amersham Biosciences, Piscataway, NJ), and molecular sizing was evaluated using the Precision protein standards (Bio-Rad).
 |
Results
|
---|
In order to identify early gene expression changes associated with liver carcinogenesis, B6C3F1 mice were exposed for 2 weeks to different compounds, including the following treatment groups: oxazepam, male and female mice, 125 (non-carcinogenic) and 2500 p.p.m. (carcinogenic); o-nitrotoluene, female mice, 1250 (non-carcinogenic) and 5000 p.p.m. (carcinogenic), and male mice, 5000 p.p.m. (100% hemangiosarcomas but no hepatocellular tumors); p-nitrotoluene, female mice, 5000 p.p.m. (non-carcinogenic); methyleugenol, female mice, 75 mg/kg (carcinogenic); eugenol, female mice, 75 mg/kg (non-carcinogenic); acetaminophen, female mice, 6000 p.p.m. (non-carcinogenic) (Table I). All of the animals were evaluated for histopathological changes. No chemically related microscopic changes were apparent in livers from the treated mice except for centrilobular hepatocellular hypertrophy in livers following treatment with the carcinogenic dose (2500 p.p.m.) of oxazepam for 2 weeks (for a detailed histopathology report see Histopath Table in online supplementary material at http://dir.niehs.nih.gov/microarray/devereux).
In a previous study (7) we identified 12 genes and ESTs that were dysregulated in the same direction in mouse liver following treatment with oxazepam and Wyeth-14,643 for 2 weeks and 6 months (7) and we hypothesized that these genes might be potential early biomarkers of mouse liver carcinogenesis. These 12 genes and ESTs included Cyp2b10, Gadd45b, Tsc-22, Lapser1, Bad, Maff, Igf-I, Igf-II, Igfbp1, Igfbp5, EST2 (GenBank accession no. AA237173) and EST3 (GenBank accession no. AA212241). Using liver RNA samples from the present study groups we examined, using quantitative real-time PCR (QRT-PCR), whether expression of these genes was changed in liver after treatment for 2 weeks with different doses of oxazepam or with the other known carcinogens or non-carcinogens (Table II). Comparing the gene expression levels between livers from male mice following treatment with 2500 p.p.m. oxazepam for 2 weeks at age 8 weeks in this study and for 2 weeks at age 7.5 months in a previous study (7), almost all of the gene expression changes induced by oxazepam treatment were consistent. A notable exception was the expression of Igfbp1, which was repressed in livers from young mice (this study) but induced in livers from mature mice (7) following treatment with oxazepam at 2500 p.p.m. Surprisingly, expression of most of these 12 genes was not significantly altered by the other treatments, including the carcinogens o-nitrotoluene and methyleugenol. The results suggest that at least these genes would not make good common early biomarkers of hepatocarcinogenesis.
View this table:
[in this window]
[in a new window]
|
Table II. Changes in expression in liver determined by QRT-PCR after treatment for 2 weeks with different known carcinogens and non-carcinogens
|
|
Next, in order to determine early gene expression changes associated with exposure to known non-genotoxic or weakly genotoxic carcinogens or to non-carcinogens, we employed the Agilent mouse oligonucleotide array, which contains >20 000 sequence verified mouse genes and ESTs. We were able to reduce the probability of false positives in our data set by performing duplicate hybridizations with fluor reversals on four independent liver samples from each treatment group compared with RNA from a pool of 10 sex-matched control livers. By analysis with the Rosetta Resolver program, we identified genes as differentially expressed if they were increased or decreased 1.5-fold or more in the same direction with a p value
0.05 in three or four out of four animals (using two chips per animal) in at least one treatment group. A total of 421 genes met these criteria and these genes were utilized for further comparative analyses (e.g. GeneSpring and Ingenuity). The entire data set is available at http://dir.niehs.nih.gov/microarray/devereux. Because of cost and time considerations, microarray analysis was not performed on livers from 125 p.p.m. oxazepam-treated or acetaminophen-treated mice. Gene expression levels in livers from these treatment groups were assessed by QRT-PCR only. Since we were interested in gene expression changes associated with carcinogen treatment, we used these non-carcinogens in addition to p-nitrotoluene and eugenol for comparisons.
When the microarray data from the eight treatment groups were clustered into a condition tree by Gene Spring analysis in a standard correlation using the average of the four animals per group for the 421 signature genes, the relationship of one treatment group to another was as expected (Figure 1a). The male and female oxazepam-treated groups clustered together, as did the non-carcinogenic p-nitrotoluene and low dose o-nitrotoluene groups, the high dose male and female o-nitrotoluene-treated mice and the methyleugenol-treated and eugenol-treated groups. These data suggest that there are patterns of early gene expression changes that distinguish these different carcinogen treatment groups from each other, as well as the carcinogens from the non-carcinogens. However, when the individual animals were clustered in a similar manner (Figure 1b; see also online supplementary material at http://dir.niehs.nih.gov/microarray/devereux), the clustering was not as clear by treatment group. All four animals per group clustered tightly into the oxazepam-, p-nitrotoluene-, low dose o-nitrotoluene- and methyleugenol-treated groups, whereas the individuals in the high dose o-nitrotoluene male and female groups and the eugenol treatment groups were not tightly clustered. These data indicate the importance of using multiple samples to obtain a good representation of the likely predicted gene expression changes due to each carcinogen at early time points. Possibly the short exposure time during a period when the liver is actively growing may account in part for the individual variability. Our focus for this report was to make an overall assessment of the differences among chemical treatment groups and not examine in detail the variation between individuals.

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 1. Clustering of 421 signature genes from oligonucleotide microarray data by chemical using the Condition Tree algorithm in Gene Spring 6.1.1. Signature genes are genes increased or decreased 1.5-fold in the same direction with a P value of 0.05 in 3 or 4 of 4 animals in at least one treatment group. OX, oxazepam; ONT, o-nitrotoluene; PNT, p-nitrotoluene; MEUG, methyleugenol; EUG, eugenol; M, male; F, female. Numbers represent the dose groups.
|
|
The number of altered genes as determined by oligonucleotide array analysis in the livers from different chemical treatments varied with the type of chemical exposure and dose. Expression of 221 genes in males or 183 genes in females was changed in liver following treatment with the 2500 p.p.m. carcinogenic dose of oxazepam. Among them we observed the up- or down-regulation of many cytochrome P450 genes, such as Cyp4a10, Cyp4a14, Cyp2b20, Cyp2c40 and others, and
60 other metabolism-associated genes, including glutathione S-transferases, NAD(P)H oxidoreductase 1 (Nqo1), epoxide hydrolase, aldehyde dehydrogenase, lipid metabolism genes and others (data not shown). The changes in metabolism-associated gene expression levels were comparable to our previous cDNA microarray results and were discussed in that manuscript (7). In contrast, few of these metabolism-associated genes were altered by the other carcinogens and non-carcinogens and, thus, smaller numbers of gene expression changes were detected for the other chemical groups. The number of genes with altered expression in livers from the 5000 p.p.m. o-nitrotoluene treatment group was similar between males and females, 76 and 74, respectively. Expression changes occurred in only 26 and 33 genes in livers from the low dose (1250 p.p.m.) o-nitrotoluene-treated and 5000 p.p.m. p-nitrotoluene-treated female mice, respectively. We identified 47 genes with altered expression in livers from the methyleugenol treatment group, while the number of altered genes in livers treated with its non-carcinogenic congener eugenol was only 22. These data demonstrate that there were more gene expression changes in livers of the carcinogen-treated mice than the non-carcinogen groups, both considering all groups and the structurally similar carcinogens and non-carcinogens, i.e. o-nitrotoluene versus p-nitrotoluene and methlyeugenol versus eugenol.
To validate the microarray data, to provide more quantitative data and to expand the investigation to other treatment groups, we performed QRTPCR on 12 genes, including two genes (Tsc-22 and Gadd45b) shown in Table II. We tested each sample in duplicate, using three livers from each compound treatment group. These genes were chosen based on their changes in expression in livers from at least one of the carcinogen groups, their potential roles in carcinogenesis and our interest for further studies. As shown in Table III, the QRT-PCR measurements in general correlated positively with the microarray data, although the QRT-PCR results sometimes showed greater differences. This may be due at least partly to differences in methodology (17). We treated the microarray data as a screening tool and the QRT-PCR measurements as a more accurate assessment of expression levels.
View this table:
[in this window]
[in a new window]
|
Table III. Changes in expression in livers by oligo array and QRT-PCR after treatment for 2 weeks with different known carcinogens and non-carcinogens
|
|
There were some notable changes in expression that occurred almost exclusively in livers from some of the carcinogen-treated mice and are likely associated with the early carcinogenic process. Foremost was loss of expression of the fragile histidine triad gene (Fhit) and the WW domain-containing oxidoreductase (Wwox) gene only in livers from the o-nitrotoluene- and methyleugenol-treated mice. Suppression of Fhit and Wwox by o-nitrotoluene was dose dependent (Table III and Figure 2). These genes are putative human tumor suppressor genes and are often lost together in many human cancers (1820). Other changes that may play a role early in hepatocarcinogenesis included strong up-regulation of growth arrest and DNA-damage-inducible 45ß (Gadd45b) and down-regulation of transforming growth factor ß stimulated clone 22 (Tsc-22), a transcriptional repressor and putative tumor suppressor gene (21,22), in livers from the carcinogenic oxazepam treatment groups. Strong up-regulation of the cell cycle genes cyclin G1 (Ccng1) and p21 (Cdkn1a) occurred in livers from the o-nitrotoluene- and methyleugenol-treated groups and down-regulation of the epidermal growth factor receptor (Egfr) in livers from the carcinogenic oxazepam- and o-nitrotoluene-treated groups. The number of genes with changes in expression in livers from mice treated with non-carcinogenic chemicals, including 1250 p.p.m. o-nitrotoluene or p-nitrotoluene, was small and we confirmed a doseresponse relationship for eight genes by QRT-PCR for the high and low doses of oxazepam and six genes for the low and high doses of o-nitrotoluene (Figure 2 and Table III).

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 2. Fold changes in expression of eight genes in liver after 2 weeks of chemical treatment determined by QRT-PCR, grouped to show doseresponse relationships and structurally similar known carcinogens versus non-carcinogens. Log scale shown. These data were derived from Table 3 and show means ± SE for three individual samples in replicate compared with a sex-matched control pool of 10 livers.
|
|
The transcription factor early growth response 1 (Egr1) was down-regulated in liver after treatment with carcinogenic doses of oxazepam in male and female mice and following o-nitrotoluene at 5000 p.p.m. in males, whereas up-regulation was found in the liver following treatment with methyleugenol or p-nitrotoluene. Inhba, a member of the transforming growth factor ß superfamily, and Junb, a negative regulator of proliferation genes, were down-regulated in livers from all groups except for the 125 p.p.m. oxazepam- and acetaminophen-treated mice. Junb gene was up-regulated as determined by QRT-PCR in liver following treatment with o-nitrotoluene, p-nitrotoluene and methyleugenol at 2 weeks (Table III). The gene that codes for cytokine inducible SH2-containing protein (Cish) is a member of a family of intracellular proteins that regulates the response of immune cells to cytokines. Cish was down-regulated in livers from all treatment groups except for the low dose o-nitrotoluene group. DNase II
(Dnase2a) encodes one of many endonucleases implicated in DNA digestion during apoptosis, and it was repressed in the liver following treatment of female mice with o-nitrotoluene.
Western blot analysis was also performed to further validate and expand on the oligonucleotide microarray analysis. Expression of Egfr, Fhit and Wwox proteins was evaluated for two liver samples from each treatment group. As shown in Figure 3, protein expression of Fhit and Wwox correlated well with the gene expression levels for all chemical groups examined. These proteins were strongly down-regulated in liver following 2 weeks treatment with methyleugenol or o-nitrotoluene, whereas they were not changed in the liver after oxazepam, p-nitrotoluene, eugenol or acetaminophen treatment. Egfr protein expression was repressed in livers from oxazepam- and o-nitrotoluene-treated mice, although there was a lower expression in livers from control females compared with males and some inter-animal variation within treatment groups (Figure 3).

View larger version (56K):
[in this window]
[in a new window]
|
Fig. 3. Protein expression of Egfr, Fhit and Wwox in liver after 2 weeks treatment with different known carcinogens and non-carcinogens. Actin is shown as a protein loading control.
|
|
Genespring and GenMapp2.0 analyses were utilized to classify the dysregulated genes into functional pathways. One category of genes that seemed to be generally affected in liver by the carcinogens in this study at 2 weeks after treatment was the cell cycle-related genes (Figure 4). Among the 421 signature genes were at least 10 cell cycle-related genes, Gadd45b, Ect2, Sipa1, Ccng1, Gas1, Mcm3, Cdkn1a, G0s2, Junb and Egfr, according to the Gene Spring ontology categories. The expression of each individual gene was altered by only one or a few of the treatments. However, the overall pattern of these genes showed large changes among the carcinogens oxazepam, 5000 p.p.m. o-nitrotoluene and methyleugenol and small or no changes among the non-carcinogens 1250 p.p.m. o-nitrotoluene, p-nitrotoluene and eugenol. These data suggest that some of the earliest changes in the carcinogenic process involve perturbations of the cell cycle, although the individual genes involved may differ among carcinogens. In addition, the Gene Spring analyses identified many changes in gene expression in metabolic pathways in the liver from oxazepam-treated mice. However, signal transduction and other cancer-associated pathways did not appear to be significantly affected by 2 weeks treatment with any of the chemicals.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 4. Changes in expression of 10 cell cycle-related genes in livers from mice treated with different known carcinogens or non-carcinogens. Linear scale of normalized intensity change compared with expression in control livers.
|
|
 |
Discussion
|
---|
A major goal of this study was to gain a better understanding of the mechanisms of the early stages of mouse liver carcinogenesis. One of the most interesting findings was the striking difference in early expression patterns of cancer-related genes in the liver associated with oxazepam treatment compared with those of methyleugenol and o-nitrotoluene (Figure 5), since these chemicals seemed to induce a similar strong tumor response in mice. In NTP 2 year studies the 2500 p.p.m. dose of oxazepam and the 75 mg/kg dose of methyleugenol (as used in this study) induced hepatocellular neoplasms in 100% of the mice, in addition to a high incidence of hepatoblastomas, a rare tumor in untreated B6C3F1 mice (9,11). The liver tumors induced by these two compounds appeared histologically similar and had similar molecular alterations, including a high frequency of ß-catenin mutations, increased Wnt signaling and cyclin D1 up-regulation, but no H-ras mutations (2,6,10). The transcriptional repressor and putative tumor suppressor Tsc-22 was down-regulated in liver tumors induced by both of these compounds (unpublished data). Also, after 2 weeks treatment with these chemicals minimal histological changes, except for moderate centrilobular hypertrophy associated with oxazepam treatment, were detected in the liver. All these results suggested that the molecular alterations and possibly the pathogenesis would be similar in the early development of hepatocarcinogenesis induced by oxazepam and methyleugenol (Figure 5). However, after 2 weeks treatment with these two compounds only 12 common genes with significantly changed expression were identified (data not shown). In addition, these were genes for which we did not find obvious associations with the carcinogenic process.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 5. Scheme of gene expression changes during the early carcinogenic process and in liver tumors after treatment with oxazepam, methyleugenol and o-nitrotoluene. Induced genes are represented in black with up arrows and repressed genes are shown in gray with down arrows. E and P in parentheses show those genes with similarly changed expression by the non-carcinogens eugenol and p-nitrotoluene.
|
|
Expression changes were identified in 183 genes in livers from the mice treated for 2 weeks with oxazepam and 47 genes in livers from the methyleugenol-treated mice. Oxazepam induced many genes in the liver associated with phase I and II xenobiotic metabolism, while methyleugenol affected expression of these genes only minimally, if at all. In fact, oxazepam was the only chemical studied that induced a large number of cytochrome P-450 enzymes, such as the Cyp2b family, which produces oxidative stress through generation of superoxide radicals (23,24). It is likely that oxidative damage caused by the induced cytochrome P-450s contributes in part to the different gene expression changes observed in liver after oxazepam treatment. Thus, the xenobiotic metabolism-associated genes accounted for a large part of the difference in number of genes dysregulated by these two carcinogens.
One remarkable discovery was the reduced or absent Fhit and Wwox expression in liver following only 2 weeks treatment with o-nitrotoluene or methyleugenol, whereas these genes were not repressed by oxazepam. The function of these genes/proteins is still being determined, although evidence is accumulating for a role in apoptosis (1719). For example, in a human pancreatic cell line lacking FHIT, reintroduction of FHIT led to cell growth inhibition and apoptosis (25). Moreover, there was a strong spontaneous tumor response, including liver hemangiomas and an adenoma, in Fhit-deficient mice (26). Loss of Fhit has been detected in a variety of rodent tumors and human cancers (19), including
65% of human hepatocellular carcinomas examined in one study (27). Repression of FHIT gene expression has also been detected in several precancerous lesions or early cancers in humans (19), suggesting that it is an early event in cancer formation. Based on our analyses, Fhit and Wwox expression in liver tissues appeared to be correlated and down-regulated in a carcinogen-specific manner. A strong connection between FHIT and WWOX expression in human gastric carcinomas and in invasive breast carcinoma has also been reported recently (28,29). Our finding of almost complete loss of these proteins suggests that mouse liver after short-term treatment with carcinogens would be an excellent model in which to study Fhit and Wwox function.
Another potentially important expression change involved a gene encoding the novel transcriptional repressor Tsc-22 (22). The findings that TSC-22 was down-regulated in human salivary gland tumors (21) and up-regulated by the anticancer drug Vesnarinone in a salivary gland carcinoma cell line (30) suggest that it is a tumor suppressor gene. In our studies Tsc-22 was down-regulated in liver following treatment for 2 weeks only with carcinogenic doses of oxazepam and Wyeth 14,643 (7), but not the other carcinogens. We also found down-regulation of this gene/protein in liver tumors induced by oxazepam and other carcinogens (including methyleugenol), as well as in spontaneous tumors from 2 year carcinogenicity studies (unpublished data). These results suggest that down-regulation of Tsc-22 plays a role in the development of all mouse liver tumors and may be an early event for certain carcinogens.
Among the early changes in cells that appear necessary for development of cancer seems to be the blocking of apoptosis (31) and it has been implicated previously as a mechanism for non-genotoxic hepatocarcinogenesis in mice and rats (9). Our study indicates that many of the cancer-associated changes in gene expression at 2 weeks following chemical treatment may have a role in evasion of apoptosis. For example, recent reports have found that overexpression of the Tsc-22, Fhit or Wwox genes resulted in increased apoptosis (3234) and in our study these genes were down-regulated by the carcinogens, suggesting that loss of these genes causes decreased apoptosis. Gadd45b, which was highly up-regulated by oxazepam in our study, may have a dual or paradoxical role in apoptosis. One study reported that Gadd45b triggers apoptotic cell death in response to transforming growth factor ß (35), whereas another study demonstrated that Gadd45b suppresses tumor necrosis factor
-induced apoptosis (36). This apparent contradiction may be due to tissue-specific differences in gene expression or differences in transcriptional complex formation. In our previous study (7) we found that tumor necrosis factor
-induced protein 2 (Tnfaip2) was induced in liver after 2 weeks treatment with 2500 p.p.m. oxazepam, suggesting that oxazepam may have a similar effect as tumor necrosis factor
. DNase II
may be involved in DNA digestion during apoptosis, and its expression was decreased in liver after treatment with o-nitrotoluene. These findings suggest that acquired resistance to apoptosis may be an important early event in hepatocarcinogenesis in mice. Several of the genes mentioned above appear to be involved in both the apoptotic process and in the cell cycle. Our data indicate that each carcinogen may invoke a different mechanism or different group of proteins to block or evade apoptosis and accelerate through the cell cycle.
Comparisons of early changes in expression between the paired carcinogens and non-carcinogens, such as methyleugenol and eugenol, can aid in our understanding of the 2 year tumor response. Microarray analysis showed that 14 of the 22 genes with altered expression in liver following treatment of mice with eugenol were the same as those detected after methyleugenol treatment, although the expression changes were less pronounced in the eugenol group (Table III and Figure 2). A notable difference was the strongly repressed expression of Fhit and Wwox by the carcinogen methyleugenol but no change for the eugenol treatment group; its significance has been discussed above. The NTP reported that there was only equivocal evidence that eugenol was a hepatocarcinogen based on its 2 year carcinogenicity study (11), which may be due to more efficient metabolism of this analog. Based on our data for these chemicals at the same treatment doses, the effect of eugenol on liver carcinogenesis may be similar to that of a low dose of methyleugenol.
Comparisons of gene expression changes were also made for o-nitrotoluene and its non-carcinogenic isomer p-nitrotoluene. After 2 weeks treatment of female mice with o-nitrotoluene, 76 gene expression changes were detected in the liver, while there were only 33 changes after p-nitrotoluene; only 17 of the changes were common. Of the 12 genes examined in Table III that we found to have potential effects on carcinogenesis, seven showed significantly different expression patterns after treatment with these two nitrotoluenes. Differences in metabolism of these nitrotoluenes to reactive intermediates may be responsible for the differences observed in their carcinogenicity (37).
We also compared gene expression changes in liver after treatment with 5000 p.p.m. o-nitrotoluene between males and females, since the NTP bioassay found clear evidence of liver tumors in females but not in males (10). We hypothesized that the hepatocellular tumor response to o-nitrotoluene in males may have been obscured by the formation of hemangiosarcomas, which resulted in early death. Of the 7476 gene changes in liver detected in males and females, only 20 were common. However, among these genes were Fhit, Wwox, Dnase2a and Cish, all of which have a role in promoting apoptosis and were down-regulated in livers of both males and females by o-nitrotoluene. This suggests that the early response to this carcinogen in males and females may involve similar cancer-associated pathways. On the other hand, p-nitrotoluene is a structurally similar non-carcinogen, and of these four genes, only Cish was significantly down-regulated in livers after treatment for 2 weeks with this compound. Thus, the use of these structurally similar carcinogens and non-carcinogens is helping to delineate what early gene expression changes may be important for the cancer process.
Doseresponse relationships for some changes in expression after 2 weeks treatment with high and low doses of oxazepam and o-nitrotoluene were observed. Compared with control liver, the 125 p.p.m. low dose of oxazepam and 1250 p.p.m. low dose of o-nitrotoluene showed limited but measurable effects after 2 weeks of treatment, even though these chemicals were considered non-carcinogens. These results suggest that there is a continuum of effect, as opposed to a threshold, and that if the animal numbers were larger in the 2 year studies or in lifespan studies these doses might also be found to be carcinogenic.
In this study we have presented clear evidence that changes in the liver following treatment with carcinogens but not with related non-carcinogens occur as early as 2 weeks for some cancer-associated genes. It appears that some of the earliest events in the carcinogenic process may involve perturbations in the cell cycle and evasion of apoptosis, although these may involve different genes or groups of genes, depending on the carcinogen.
 |
Notes
|
---|
5 Present address: Department of Pathology, Biosafety Research Center, Foods, Drugs and Pesticides, 582-2 Arahama, Shioshinden, Fukude-cho, Iwata-gun, Shizuoka 437-1213, Japan 
 |
Acknowledgments
|
---|
The authors would like to thank Drs Alexandra Heinloth and Gary Boorman for their critical reading and helpful comments on the paper. Also, they acknowledge the excellent support of the NIEHS Microarray Core, especially Danica Ducharme, who did the hybridizations, and excellent technical assistance with the mouse necropsies from Mike Snell.
 |
References
|
---|
- National Toxicology Program (2002) Report on Carcinogens, 10th Edn, Carcinogen Profiles 2002. Research Triangle Park, NC.
- Anna,C.H., Sills,R.C., Foley,J.F., Stockton,P.S., Ton,T.V. and Devereux,T.R. (2000) Beta-catenin mutations and protein accumulation in all hepatoblastomas examined from B6C3F1 mice treated with anthraquinone or oxazepam. Cancer Res., 60, 28642868.[Abstract/Free Full Text]
- Anna,C.H., Iida,M., Sills,R.C. and Devereux,T.R. (2003) Expression of potential beta-catenin targets, cyclin D1, c-Jun, c-Myc, E-cadherin and EGFR in chemically induced hepatocellular neoplasms from B6C3F1 mice. Toxicol. Appl. Pharmacol., 190, 135145.[CrossRef][ISI][Medline]
- Cherkaoui-Malki,M., Meyer,K., Cao,W.Q., Latruffe,N., Yeldandi,A.V., Rao,M.S., Bradfield,C.A. and Reddy,J.K. (2001) Identification of novel peroxisome proliferator-activated receptor alpha (PPARalpha) target genes in mouse liver using cDNA microarray analysis. Gene Expr., 9, 291304.[ISI][Medline]
- Christensen,J.G., Romach,E.H., Healy,L.N. et al. (1999) Altered bcl-2 family expression during non-genotoxic hepatocarcinogenesis in mice. Carcinogenesis, 20, 15831590.[Abstract/Free Full Text]
- Devereux,T.R., Anna,C.H., Foley,J.F., White,C.M., Sills,R.C. and Barrett,J.C. (1999) Mutation of beta-catenin is an early event in chemically induced mouse hepatocellular carcinogenesis. Oncogene, 18, 47264733.[CrossRef][ISI][Medline]
- Iida,M., Anna,C.H., Hartis,J. et al. (2003) Changes in global gene and protein expression during early mouse liver carcinogenesis induced by non-genotoxic model carcinogens oxazepam and Wyeth-14,643. Carcinogenesis, 24, 757770.[Abstract/Free Full Text]
- Gonzales,A.J., Christensen,J.G., Preston,R.J., Goldsworthy,T.L., Tlsty,T.D. and Fox,T.R. (1998) Attenuation of G1 checkpoint function by the non-genotoxic carcinogen phenobarbital. Carcinogenesis, 19, 11731183.[Abstract]
- Christensen,J.G., Gonzales,A.J., Cattley,R.C. and Goldsworthy,T.L. (1998) Regulation of apoptosis in mouse hepatocytes and alteration of apoptosis by nongenotoxic carcinogens. Cell Growth Differ., 9, 815825.[Abstract]
- Devereux,T.R., White,C.M., Sills,R.C., Bucher,J.R., Maronpot,R.R. and Anderson,M.W. (1994) Low frequency of H-ras mutations in hepatocellular adenomas and carcinomas and in hepatoblastomas from B6C3F1 mice exposed to oxazepam in the diet. Carcinogenesis, 15, 10831087.[Abstract]
- NTP (1993) Toxicology and Carcinogenesis Studies of Oxazepam (CAS No. 604-75-1) in Swiss-Webster and B6C3F1 Mice (Feed Studies), National Toxicology Program Technical Report Series, Vol. 443. NTP, Research Triangle Park, NC.
- NTP (2002) Toxicology and Carcinogenesis Studies of o-Nitrotoluene (CAS No. 88-72-2) in F344/N Rats and B6C3F1 Mice (Feed Study), National Toxicology Program Technical Report Series, Vol. 504. NTP, Research Triangle Park, NC.
- NTP (2002) Toxicology and Carcinogenesis Studies of p-Nitrotoluene (CAS No. 99-99-0) in F344/N Rats and B6C3F1 Mice (Feed Studies), National Toxicology Program Technical Report Series, Vol. 498. NTP, Research Triangle Park, NC.
- NTP (2000) Toxicology and Carcinogenesis Studies of Methyleugenol (CAS No. 93-15-2) in F344/N Rats and B6C3F1 Mice (Gavage Studies), National Toxicology Program Technical Report Series, Vol. 491. NTP, Research Triangle Park, NC.
- Burkey,J.L., Sauer,J.M., McQueen,C.A. and Sipes,I.G. (2000) Cytotoxicity and genotoxicity of methyleugenol and related congenersa mechanism of activation for methyleugenol. Mutat. Res., 453, 2533.[ISI][Medline]
- NTP (1993) Toxicology and Carcinogenesis Studies of Acetaminophen (CAS No. 103-90-2) in F344 Rats and B6C3F1 Mice (Feed Studies), National Toxicology Program Technical Report Series, Vol. 394. NTP, Research Triangle Park, NC.
- Yuen,T., Wurmbach,E., Pfeffer,R.L., Ebersole,B.J. and Sealfon,S.C. (2002) Accuracy and calibration of commercial oligonucleotide and custom cDNA microarrays. Nucleic Acids Res., 30, e48.[Abstract/Free Full Text]
- Ludes-Meyers,J.H., Bednarek,A.K., Popescu,N.C., Bedford,M. and Aldaz,C.M. (2003) WWOX, the common chromosomal fragile site, FRA16D, cancer gene. Cytogenet. Genome Res., 100, 101110.[ISI][Medline]
- Ishii,H., Ozawa,K. and Furukawa,Y. (2003) Alteration of the fragile histidine triad gene early in carcinogenesis: an update. J. Exp. Ther. Oncol., 3, 291296.[CrossRef][Medline]
- Pekarsky,Y., Zanesi,N., Palamarchuk,A., Huebner,K. and Croce,C.M. (2002) FHIT: from gene discovery to cancer treatment and prevention. Lancet Oncol., 3, 748754.[CrossRef][ISI][Medline]
- Nakashiro,K., Kawamata,H., Hino,S., Uchida,D., Miwa,Y., Hamano,H., Omotehara,F., Yoshida,H. and Sato,M. (1998) Down-regulation of TSC-22 (transforming growth factor beta-stimulated clone 22) markedly enhances the growth of a human salivary gland cancer cell line in vitro and in vivo. Cancer Res., 58, 549555.[Abstract]
- Shibanuma,M., Kuroki,T. and Nose,K. (1992) Isolation of a gene encoding a putative leucine zipper structure that is induced by transforming growth factor beta 1 and other growth factors. J. Biol. Chem., 267, 1021910224.[Abstract/Free Full Text]
- Griffin,R.J., Dudley,C.N. and Cunningham,M.L. (1996) Biochemical effects of the mouse hepatocarcinogen oxazepam: similarities to phenobarbital. Fundam. Appl. Toxicol., 29, 147154.[CrossRef][ISI][Medline]
- Waxman,D.J. and Azaroff,L. (1992) Phenobarbital induction of cytochrome P-450 gene expression. Biochem. J., 281, 577592.[ISI][Medline]
- Dumon,K.R., Ishii,H., Vecchione,A. et al. (2001) Fragile histidine triad expression delays tumor develpment and induces apoptosis in human pancreatic cancer. Cancer Res., 61, 48274836.[Abstract/Free Full Text]
- Zanesi,N., Fidanza,V., Fong,L.Y. et al. (2001) The tumor spectrum in FHIT-deficient mice. Proc. Natl Acad. Sci. USA, 98, 1025010255.[Abstract/Free Full Text]
- Zhao,P., Song,X., Nin,Y., Lu,Y. and Li,X. (2003) Loss of fragile histidine triad protein in human hepatocellular carcinoma. World J. Gastroenterol., 9, 12161219.[ISI][Medline]
- Aqeilan,R.I., Kuroki,T., Pekarsky,Y., Albagha,O., Trapasso,F., Baffa,R., Huebner,K., Edmonds,P. and Croce,C.M. (2004) Loss of WWOX expression in gastric carcinoma. Clin. Cancer Res., 10, 30533058.[Abstract/Free Full Text]
- Guler,G., Uner,A., Guler,N., Han,S.Y., Iliopoulos,D., Hauck,W.W., McCue,P. and Huebner,K. (2004) The fragile genes FHIT and WWOX are inactivated coordinately in invasive breast carcinoma. Cancer, 100, 16051614.[CrossRef][ISI][Medline]
- Kawamata,H., Nakashiro,K., Uchida,D., Hino,S., Omotehara,F., Yoshida,H. and Sato,M. (1998) Induction of TSC-22 by treatment with a new anti-cancer drug, vesnarinone, in a human salivary gland cancer cell. Br. J. Cancer, 77, 7178.[ISI][Medline]
- Hanahan,D. and Weinberg,R.A. (2000) The hallmarks of cancer. Cell, 100, 5770.[ISI][Medline]
- Uchida,D., Kawamata,H., Omotehara,F., Miwa,Y., Hino,S., Begum,N.M., Yoshida,H. and Sato,M. (2000) Over-expression of TSC-22 (TGF-beta stimulated clone-22) markedly enhances 5-fluorouracil-induced apoptosis in a human salivary gland cancer cell line. Lab. Invest., 80, 955963.[ISI][Medline]
- Omotehara,F., Uchida,D., Hino,S., Begum,N.M., Yoshida,H., Sato,M. and Kawamata,H. (2000) In vivo enhancement of chemosensitivity of human salivary gland cancer cells by overexpression of TGF-beta stimulated clone-22. Oncol. Rep., 7, 737740.[ISI][Medline]
- Chang,N.S., Doherty,J., Ensign,A., Lewis,J., Heath,J., Schultz,L., Chen,S.T. and Oppermann,U. (2003) Molecular mechanisms underlying WOX1 activation during apoptotic and stress responses. Biochem. Pharmacol., 66, 13471354.[CrossRef][ISI][Medline]
- Yoo,J., Ghiassi,M., Jirmanova,L., Galliet,A.G., Hoffman,B., Fornace,A.J.,Jr, Liebermann,D.A., Bottinger,E.P. and Roberts,A.B. (2003) Transforming growth factor-beta-induced apoptosis is mediated by Smad-dependent expression of GADD45b through p38 activation. J. Biol. Chem., 278, 4300143007.[Abstract/Free Full Text]
- Papa,S., Zazzeroni,F., Bubici,C. et al. (2004) Gadd45 beta mediates the NF-kappa B suppression of JNK signalling by targeting MKK7/JNKK2. Nature Cell Biol., 6, 146153.[CrossRef][ISI][Medline]
- Dunnick,J., Burka,L.T., Mahler,J. and Sills,R. (2003) Carcinogenic potential of o-nitrotoluene and p-nitrotoluene. Toxicology, 183, 221234.[CrossRef][ISI][Medline]
Received September 17, 2004;
revised December 8, 2004;
accepted December 10, 2004.