Genomic analysis of alachlor-induced oncogenesis in rat olfactory mucosa
Mary Beth Genter1,
Dawn M. Burman1,
Soundarapandian Vijayakumar2,
Cathy L. Ebert3 and
Bruce J. Aronow3
1 Departmet of Environmental Health, University of Cincinnati, Cincinnati, Ohio 45267-0056
2 Department of Medicine, Columbia University, New York, New York 10032
3 Childrens Hospital Medical Center, Cincinnati, Ohio 45229
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ABSTRACT
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Alachlor induces olfactory mucosal tumors in rats in a highly ordered temporal process. We used GeneChip analysis to test the hypothesis that histological progression and oncogenic transformation are accompanied by gene expression changes that might yield clues as to the molecular pathogenesis of tumor formation. Acute alachlor exposure caused upregulation of matrix metalloproteinases (MMP)-2 and -9, tissue inhibitor of metalloproteinase-1, carboxypeptidase Z, and other genes related to extracellular matrix homeostasis. Heme oxygenase was upregulated acutely and maintained elevated expression. Expression of ebnerin, related to the putative human tumor suppressor gene DMBT1, progressively increased in alachlor-treated olfactory mucosa. Progression from adenomas to adenocarcinoma was correlated with upregulation of genes in the wnt signaling pathway. Activated wnt signaling was confirmed by immunohistochemical localization of ß-catenin to nuclei of adenocarcinomas, but not earlier lesions. These observations suggest that initiation and progression of alachlor-induced olfactory mucosal tumors is associated with alterations in extracellular matrix components, induction of oxidative stress, upregulation of ebnerin, and final transformation to a malignant state by wnt pathway activation.
gene expression; carcinogenesis; ß-catenin
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INTRODUCTION
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ALACHLOR [2-chloro-2',6'-diethyl-N-(methoxymethyl)acetanilide; CAS 15972-60-8] is one of several chloracetanilide herbicides in use world-wide in the production of crops such as corn, soybeans, peanuts, and rice. The chloracetanilides are multi-site carcinogens in rodents, with olfactory mucosa, liver, thyroid, and stomach comprising the sites of carcinogenic activity in rats (72). Epidemiological evaluation of a small cohort of humans exposed simultaneously to alachlor in the manufacturing process and via highly contaminated drinking water at the manufacturing plant revealed a slightly elevated risk of colorectal cancer in this population (40). Contamination of drinking water supplies likely represents the major route of exposure for human populations, as chloracetanilide herbicides and their degradation products are frequently detected as drinking water contaminants (e.g., 8, 13).
The mechanism of alachlor-induced olfactory mucosal mutagenicity (77) and carcinogenicity is proposed to involve metabolism via several intermediate steps, which can be catalyzed in part by human cytochrome P-450 (CYP) 3A4, to 2,6-diethylaniline (14). Conversion of 2,6-diethylaniline to a quinoneimine metabolite has been demonstrated (23); quinoneimines deplete cellular antioxidants (66), and mutagenicity could result from the subsequent oxidative stress, as has been previously demonstrated (e.g., 2, 22, 30, 31, 35, 36, 54, 80). Alternatively, the nitrosobenzene derivative of 2,6-diethylaniline has been demonstrated to be mutagenic (38); such a metabolite (or a DNA-reactive nitrenium ion) could be produced in vivo by N-hydroxylation of 2,6-diethylaniline, followed by acetylation (29). In any case, it appears that the olfactory predominant enzyme, CYP2A3, may involved in the final bioactivation step, as the distribution of this enzyme in control rat olfactory mucosa correlates highly with the ultimate pattern of tumor formation in alachlor-treated rats (25).
Olfactory mucosal tumors arising as a result of alachlor exposure are present following 5 mo of exposure (126 mg·kg-1·day-1 in the diet); following 6 mo of exposure, 50% of the rats have one or more olfactory mucosal adenomas, increased cell proliferation, and numerous foci of respiratory metaplasia (25). Poorly differentiated, invasive adenocarcinomas were first noted following 11 mo of exposure. Rats killed following 1218 mo of exposure often display
20 tumors per rat (Genter et al., unpublished observations). To test the hypothesis that gene expression programs underlying the orderly series of histological changes (Fig. 1) that accompany alachlor-induced olfactory mucosal carcinogenesis reflect oncogenic mechanisms, we subjected a series of RNA samples from control and alachlor-treated rats to Affymetrix GeneChip arrays. Olfactory mucosal samples from rats treated with alachlor over a time course ranging from 1 day to 18 mo were characterized to identify gene expression alterations that paralleled histological changes in the tissue and the formation and progression of tumors.

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Fig. 1. Histological evolution of alachlor-induced olfactory mucosal tumors. Photomicrographs are presented at different magnifications to facilitate visualization of critical features. Normal olfactory mucosa (A) develops multiple foci of disorganized-appearing epithelium following >3 mo of exposure (B). These foci are characterized by development of apical cilia and a loss of the overall organizational pattern of the mucosa. Epithelial polyps (C) develop in these sites of epithelial alterations and progress to larger neoplasms displaying the histological characteristics of polypoid adenomas (D). Invasive tumors were noted following 11 mo of exposure; the tumor seen here (E) fills nearly one-half of the nasal cavity [a frontal section is shown with the nasal septum (S) labeled for orientation] and is invading the lateral wall of the nasal cavity. Bars in A and B = 80 µm; in C = 170 µm; in D = 0.5 mm; and in E = 1.0 mm.
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METHODS
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Animals, tissue samples, and RNA preparation.
Alachlor (Chem Service, West Chester, PA) was administered to male Long-Evans rats (Harlan, Indianapolis, IN) in Teklad (Harlan) powdered diet (dietary equivalent of 126 mg·kg-1· day-1) for durations ranging from 1 day to 18 mo as previously described (25). Control rats were maintained on powdered diet. Studies were approved by the University of Cincinnati Institutional Animal Care and Use Committee. Rats were killed by a pentobarbital overdose followed by decapitation according to institutional and national animal care guidelines. Ethmoid turbinates were rapidly dissected and frozen in liquid nitrogen. Frozen tissues were homogenized using a hand-held tissue grinder in TRI Reagent (MRC, Cincinnati, OH), following the manufacturers protocol. Total RNA was precipitated with ethanol/sodium acetate, resuspended in DEPC-treated water and screened for quality RNA using an Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA); acceptable samples had cutoff values of ratios of 1.8 for the 28S:18S ribosomal subunits. Each RNA sample was derived from a single rat. Two treated animals were killed, and two separate olfactory mucosal RNA samples from each were labeled and hybridized for each of the following time points: control, 3 mo, 4 mo, and 5 mo. For one of the 1-mo-treated rats, as well as the rats administered alachlor for 1 day or 4 days, a single RNA sample was labeled and hybridized (the second 1-mo-treated sample was labeled and hybridized in duplicate). For the two alachlor-induced tumors analyzed, a single RNA sample, derived from two different tumors from two different rats, was labeled and hybridized. In addition to rats killed for RNA isolation, rats dosed for the identical durations were prepared for histological observations as previously described (25), except that following fixation, tissues were decalcified in multiple changes of cold, 0.3 M EDTA (pH 7.4) prior to embedding in paraffin.
Immunohistochemistry.
Immunohistochemistry was performed on 5-µm sections prepared for histology (above). Ebnerin was localized in nasal cavity sections and alachlor-induced olfactory mucosal tumors using an anti-hensin antibody (65). This antibody also recognizes the highly related protein, intestinal crp-ductin; therefore, we used staining of intestinal sections as a positive control for the antibody staining. Visualization was achieved using an anti-guinea pig horseradish peroxidase (HRP)-conjugated secondary antibody (1:100; Dako, Carpinteria, CA) with tyramide signal amplification (TSA Biotin system; NEN Life Science Products, Boston, MA). ß-Catenin was localized with antibody from BD-Transduction Laboratories (Lexington, KY) and visualized using an HRP-conjugated anti-mouse secondary antibody and TSA amplification as described above.
Western blot analysis.
CYP2A3 levels were assessed by Western blot analysis using standard denaturing conditions (10% acrylamide mini gels) and 5 µg of olfactory mucosal microsomal protein per lane as previously described (24). Primary antibody was the gift of Dr. Xinxin Ding (New York State Department of Health, Albany, NY). Visualization was achieved using a HRP-conjugated secondary antibody (Dako), enhanced chemiluminescence (ECL reagent, Amersham), and exposure to X-ray film.
Array analysis.
Total RNA was reverse transcribed using an oligo-dT primer, followed by second-strand cDNA synthesis. T7 RNA polymerase-mediated cRNA was then biotinylated and hybridized to the Affymetrix GeneChip Rat U34A using the Affymetrix-recommended protocol (12, 50). Affymetrix MicroArray Suite version 5.0 was used to scan and quantitate Rat U34A GeneChip using default scan settings. Intensity data was scaled to target intensity of 1,500, and the results were analyzed using both MicroArray Suite 5.0 and GeneSpring 4.1.5 (Silicon Genetics, Redwood City, CA). Data values used for filtering and clustering were "signal," "signal confidence," "absolute call" (absent/present), and "change" (increase, decrease, unchanged) as implemented in MicroArray Suite 5.0. Data were normalized as follows: the 50th percentile of all measurements was used as a positive control for each array. Each measurement for each gene was divided by this synthetic positive control, assuming that this was at least 10. The bottom 10th percentile signal level was used as a test for correct background subtraction. The measurement for each gene in each sample was divided by the corresponding value in untreated samples, assuming that the value was at least 0.01. Genes regulated across the experimental study were identified by data filtering for those over- or under-expressed in at least two samples whose signal strength was greater than 500 in two samples, and were also called "present" in at least one sample. An additional approach combined those with genes that could predict length of alachlor exposure or histological response using a Kruskal-Wallis ANOVA with a cutoff value of P < 0.001 and a Benjamini-Hochberg multiple testing correction as implemented in GeneSpring. K-means analysis was similarly executed in GeneSpring to organize genes into clusters based on similar expression across the treatment time course.
Gene annotation.
Expressed sequence tags (ESTs) represented on the rat genome U34A GeneChip by Affymetrix were derived from the Rat Unigene Build no. 34 assembly. All clones represented on the chip were ESTs on gene lists of interest were subjected to re-annotation by the use of Unigene and the execution of National Center for Biotechnology Information BLASTN searches (http://www.ncbi.nlm.nih.gov/BLAST/) vs. the nonredundant nucleotide database during February-April, 2002. Gene category information was based on all publicly available gene ontology information from the Gene Ontology Consortium (http://www.geneontology.org/) as harvested from SWISS-PROT, GeneCards, Compugen, LocusLink, and GenBank as well as exhaustive Medline literature searches.
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RESULTS
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To identify alachlor-induced gene expression changes and identify separate stages in the tumorigenesis process, we performed gene expression profiling using Affymetrix RG-U34A GeneChips. The use of high-quality RNA (predictive criteria was a 28S:18S ratio of greater than 1.8 in an Agilent BioAnalyzer 2100 assay) was critical for obtaining high-quality microarray hybridization data. Successful hybridizations demonstrated a large dynamic range of cellular RNA signal values that is comparable to the range demonstrated by the spike controls and that the Affymetrix Microarray Suite 5.0 software indicated greater than 40% of genes on the U34A chip were designated present, and that the 5' to 3' ratio for GAPDH mRNA was over 0.7. Genes whose expression was affected over time by alachlor were identified from a pool of genes that fulfilled a series of initial filter criteria: 4,777 probe set elements were called "present" by the Affymetrix algorithm on at least one of 26 chips, 998 probe set elements were overexpressed by 1.8x or more in at least 2 samples, whereas 584 were underexpressed 0.5x in at least 2 of the samples. An additional approach to the detection of genes that were significantly regulated by alachlor exposure employed a Welch t-test parametric analysis of variance (ANOVA). Using a cutoff of P < 0.001 (without correcting for false discovery rate error), alachlor-exposed samples could be distinguished from untreated controls based on differential expression of 644 genes. Making a union of the under and over expressed genes with the alachlor-regulated genes and then restricting these to those that met the "present" criteria yielded 1,392 probe set elements for cluster analysis. Using the K-means algorithm, we used an empirical approach to determine that 16 sets allowed for excellent representation of the prominent patterns in the data set. Several of the clusters were highly chaotic over the sample series, and their elimination led to a list of 1,265 genes whose variance could be very well represented by using 16 K-means sets (Fig. 2). Behaviors represented within the different sets included those that were upregulated acutely; those that were upregulated only in alachlor-induced tumors (e.g., sets 1, 3, and 13); those that were downregulated with alachlor treatment (e.g., sets 8 and 16); those downregulated in alachlor-induced tumors (e.g., sets 7, 14, and 16); and those upregulated persistently across the treatment time course (e.g., sets 4, 10, and 11; Fig. 2). Alachlor treatment resulted in reduced expression of genes highly associated with differentiated, functional olfactory mucosa; e.g., the olfactory predominant cytochrome P-450 enzyme, CYP2A3, was downregulated following acute exposure, well prior to the development of histological changes (Fig. 2D; also, see Ref. 25).


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Fig. 2. Examples of alachlor-regulated genes, displayed after K-means analysis. For all graphs shown, duration of alachlor treatment increases from left to right, with 2 alachlor tumors (harvested from rats treated for 18 mo) representing the samples on the far right of the graph (see label on x-axis). Histological abnormalities are scored as follows: - -, no alachlor treatment and no histological damage observed [this applies to the 2 control (C) samples]; -, alachlor treated, but no histological changes observed (1 day, 4 days, and 1 mo samples); +, some epithelial atypia, including foci of respiratory metaplasia (3 mo and 4 mo samples); ++, more pronounced epithelial atypia and small neoplasms present in 25% of the treated rats (5 mo samples); +++, numerous tumors present, some invasive [tumor (T) samples]. Gene expression is represented along the vertical axis, with a normalized intensity value of 1.0 denoting no regulation by alachlor. A: a composite of the 16 K-means graphs. B: K-means set 3 includes 78 genes that show little regulation by alachlor except in the tumors. Representative genes of this set include mucin 1, HNF-3/Forkhead homolog-1, and pancreatitis-associated protein III. C: in contrast, K-means set 7 includes 109 genes that show little regulation except for dramatic downregulation in tumors. Representative genes of this set include homer neuronal immediate early gene, -synuclein, olfactomedin-related endoplasmic reticulum localized protein, glutamate receptor, ionotropic kainate 5, and secreted frizzled-related protein. D: K-means set 9 includes 137 genes that are acutely downregulated, display some recovery, and are then downregulated in the tumors. These include CYP2A3 [Western blot shown; 2 lanes each control olfactory mucosa, and rats treated for 2 days, 4 days, or 1 mo with alachlor prior to death (2 different rats per treatment)], CYP2F1, olfactory marker protein, insulin-like growth factor receptor, and rhodanese. E: K-means set 11 undoubtedly contains the genes that are the most highly regulated by alachlor, including the following labeled genes: Bin 1 (myc box-dependent interacting protein 1) (a); metallothionein 3 (b); and carboxypeptidase Z (c). Other genes in this set include apurinic/apyramidinic endonuclease 1 (Gadd45), tissue inhibitor of metalloproteinase-1, latexin, and DNA damage inducible transcript 1. F: K-means set 4 contains genes that are most significantly upregulated following acute (1 day to 1 mo) of alachlor exposure. Included among these genes are many associated with extracellular matrix homeostasis: procollagen C enhancer protein, collagen V 2, matrix metalloproteinase-9 (MMP-9), and MMP-2.
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Genes that were upregulated following acute alachlor exposure (1 day to 1 mo) were of particular interest because olfactory mucosal changes are not histologically evident until after 3 mo of exposure. Therefore, we identified 148 genes and ESTs whose normalized expression was upregulated 3-fold or more following 1 day, 4 days, and/or 1 mo of alachlor exposure from the 16 K-means sets (Table 1). Genes related to control of extracellular matrix formed a major subgroup of these genes, including matrix metalloproteinase-9 (MMP-9; upregulated
9-fold compared with untreated controls; MMP-2 was upregulated 2.2-fold), carboxypeptidase Z (upregulated
7-fold), and tissue inhibitor of metalloproteinase-1 (up 3-fold). Genes related to immune system function, cell proliferation/cell cycle regulation, and calcium homeostasis were also highly represented within this subgroup (Table 1). Raw normalized data, gene lists, and K-means groups can be found at http://genet.chmcc.org in the rat U34A folder under Genter et al., 2002.
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Table 1. Genes acutely upregulated 3-fold with acute (1 day to 1 mo) alachlor exposure, segregated by functional category
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Four-hundred seventeen genes and ESTs were identified within the 1,265 based on their
2-fold upregulated expression in alachlor-induced tumors relative to the untreated mucosa. Many of these genes highly expressed in the tumors could be consolidated into one of several key functional categories. Several of these are immune response genes (e.g., neutrophil defensin, mast cell protease, "squamous cell carcinoma antigen recognized by T cells," and major histocompatibility complex antigens, e.g., RT1.EC2, RT1.Doa, and RT1.Aw2), and a significant proportion are associated with cell proliferation including nucleolin [the major nucleolar protein in exponentially-growing eukaryotic cells (7)], cyclin D2, cell division cycle control protein 2, regenerating protein III, and vascular endothelial growth factor. Additional representation within the tumor-elevated gene groups included heme oxygenase 1 and glutathione synthetase, which encode proteins associated with oxidative stress responses (52). As was observed following acute alachlor exposure, many of the highly expressed tumor genes were related to extracellular matrix homeostasis [e.g., extracellular matrix protein 2 (related to SC1; Ref. 33) and fibronectin]. However, this group did not include the early response extracellular matrix modifying genes activated following acute alachlor exposure such as MMP-2 and MMP-9. Upregulation of TGFß signaling in alachlor-induced tumors is suggested by upregulation of TGFß masking protein, decorin, mothers against decapentaplegic homolog 1 (MAD-1), "ras related gene" (U12187), and N-ras. A fourth set of highly expressed tumor genes, including axin2 (Fig. 3) and frizzled, suggested activation of the wnt signaling pathway in alachlor-induced tumors (32). As activated wnt signaling results in nuclear localization of ß-catenin (discussed below), we sought to identify the occurrence of nuclear ß-catenin using immunohistochemistry in the alachlor-induced tumors as well as preneoplastic lesions. Alachlor-induced polyps and early adenomas did not display nuclear ß-catenin (not shown); however, more advanced adenocarcinomas displayed abundant cytoplasmic and nuclear ß-catenin (Fig. 3); this pattern is consistent with the observation that wnt pathway genes are not upregulated until late in the carcinogenic process. Interestingly, the staining in some tumors is heterogeneous, suggesting that there has been progression to a more malignant phenotype in portions of some of the tumors (Fig. 3).

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Fig. 3. Localization of cytoplasmic and nuclear ß-catenin in alachlor-induced olfactory mucosal tumors. Immunohistochemistry with anti ß-catenin antibody revealed heterogeneous nuclear localization (examples are identified with arrows), as well as cytoplasmic accumulation, consistent with activation of wnt signaling in alachlor-induced olfactory mucosal adenocarcinomas. Neither cytoplasmic accumulation nor nuclear localization was noted in polyps or adenomas.
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Ebnerin [the rat form of mouse crp-ductin, rabbit hensin, and human DMBT1 (46, 65)] was highly expressed after 4 mo of alachlor exposure, as well as in alachlor-induced tumors. Immunohistochemistry confirmed that the protein was readily detected in both control nasal respiratory mucosa and in alachlor-induced tumors (Fig. 4) and demonstrated the absence of ebnerin in control olfactory mucosa.

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Fig. 4. Increased ebnerin gene expression was noted in the progression of alachlor-induced olfactory mucosal alterations and in olfactory mucosal (OM) tumors. Immunohistochemistry was performed with an anti-hensin antibody, which also recognized crp-ductin, a protein localized in intestinal crypts (not shown), consistent with the known high homology between rat ebnerin and mouse crp-ductin. Note that control olfactory mucosa does not have immunohistochemically detectable ebnerin, whereas it is readily detectable on the surface of respiratory mucosal (R) cells (red reaction product). Olfactory mucosal tumors display ebnerin both on their surfaces and in the ductal lumens within the tumor; the former is consistent with the observation that the surface of alachlor-induced olfactory mucosal tumors often displays features of respiratory mucosa, e.g., apical cilia (25); the latter observation is consistent with the sequence-derived data suggesting that ebnerin is a secreted protein (41).
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DISCUSSION
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As predicted, the histological changes accompanying the formation of alachlor-induced olfactory mucosal tumors were accompanied by both early and late changes in gene expression. Early upregulation of multiple extracellular matrix genes, particularly metalloproteinases, is not unique to alachlor-induced olfactory mucosal carcinogenesis. Matrilysin (MMP-7) is important in the early stages of intestinal tumorigenesis, and ablating the gene significantly reduced tumor formation in two different animal models of intestinal tumorigenesis (15, 26, 78). In addition, the broad-spectrum metalloproteinase inhibitor batimastat greatly decreased the incidence of intestinal tumors in mice heterozygous for the adenomatous polyposis coli (Apc) tumor suppressor gene (26). These observations suggest that inhibition of the initial burst of metalloproteinase activity following acute alachlor exposure might reduce or eliminate the olfactory mucosal carcinogenicity of this compound. MMP-2 and MMP-9 are not only associated with alachlor-induced olfactory mucosal tumors, but are also associated with neovascularization and tumor progression in human and murine breast cancers (6, 17, 34).
The observation that axin and frizzled genes were highly upregulated in alachlor-induced tumors strongly suggests that alachlor treatment activates wnt pathway signaling. Other important genes/proteins in this pathway include ß-catenin, Apc, glycogen synthetase kinase ß, and disheveled (27). Wnt/ß-catenin signaling is frequently activated in epithelial-derived cancer cells by stabilizing mutations of ß-catenin or loss-of-function mutations of the Apc tumor suppressor gene (76). ß-Catenin is involved in regulation of cell adhesion and epithelial cell differentiation (19, 44). Individuals who inherit mutations or deletions of the Apc tumor suppressor gene develop hundreds to thousands of colonic polyps (familial adenomatous polyposis) (27). Mutations in Apc generally result in the formation of a truncated protein product that is ineffective in regulating cytoplasmic levels of ß-catenin (27). Accumulation of cytoplasmic ß-catenin results in interactions between ß-catenin and the cytoplasmic Tcf transcription factors, nuclear translocation of the ß-catenin/Tcf complex, and the transcriptional activation of genes such as c-myc, c-jun, fra-1, and cyclin D1 (37). Nuclear translocation of ß-catenin occurs in familial adenomatous polyposis, and we have demonstrated that nuclear translocation of ß-catenin occurs in alachlor-induced olfactory mucosal tumors (Fig. 3). Therefore, we hypothesize that alachlor-induced olfactory mucosal carcinogenesis may involve activation of the wnt signaling pathway.
There is precedence for toxicant-induced mutations in Apc. N-nitrosobis(2-hydroxypropyl)amine (BHP)-induced mutations in Apc were detected in rat lung adenomas and adenocarcinomas, but not in hyperplastic epithelial lesions (70). BHP is, in addition, associated with mutations of ß-catenin phosphorylation sites in the same model system (70). In contrast, the carcinogenic amine 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine, associated with cooked meats, caused Apc mutations with as little as 1 wk of exposure in rats (10). In our experiments, nuclear localization of ß-catenin appeared to be associated with the progression, rather than the initiation, of alachlor-induced olfactory neoplasms, as nuclear localization of ß-catenin was not found in early lesions.
Alachlor is mutagenic in the presence of an olfactory mucosal activating system (77), but the target gene(s) in the rat have not been determined. Alachlor causes marked perturbations in olfactory mucosal antioxidants, which could potentially be associated with oxidative DNA damage (9). Oxidative DNA damage causes point mutations and is associated with subsequent tumor formation in vivo (2, 22, 30, 31, 36). Following acute alachlor exposure, we observed the approximate twofold upregulation of multiple genes that are classically associated with oxidative stress responses, namely heme oxygenase, glutathione synthase, and metallothionein (MT)-1 and MT-2 (3, 52). Interestingly, MT-3 was also very highly expressed acutely and in alachlor-induced tumors (Fig. 2). In normal tissues, MT-3 expression has only been reported in brain and deciduum, and is not upregulated by stress (11, 18, 71). Several human tumors, including breast and bladder, have recently been reported to express MT-3 (59, 60). Despite its dramatic upregulation in alachlor-treated olfactory mucosa, it is unclear what role MT-3 may play in the carcinogenic process.
Genes encoding pancreatitis-associated proteins (PAP) I and III, together with mucin 1, were also significantly upregulated in alachlor-induced olfactory mucosal tumors. PAP I is a secretory stress protein that was first described in acute pancreatitis (73), but PAPs I and III are constitutively expressed and coordinately regulated in intestine (57). PAP I modified the adhesion and motility of melanocytes in vitro, suggesting a potential role in melanoma invasion (73). PAP I has anti-apoptotic and mitogenic properties (5) and is expressed in heptoblastomas (21). Mucin 1 is frequently detected in human head and neck cancers (16) and was observed in invasive mucinous cystic pancreatic neoplasms, but not in noninvasive neoplasms, suggesting that mucin 1 expression may represent a marker of progression from a noninvasive to an invasive tumor type (42).
Growth factors of the TGFß family are potent regulators of epithelial proliferation, differentiation, and extracellular matrix regulation, with TGFß generally regarded as a growth-inhibitory cytokine (e.g., 58, 75). It has been proposed that dysregulation of TGFß is pivotal in many carcinogenic processes, likely involving MAP kinase and AP-1 signaling pathways (55); both MAP kinase kinase (MAP2K) and MAP3K were among genes upregulated by acute alachlor exposure and in alachlor-induced tumors. Two primary signaling cascades downstream of the TGFß receptors have been elucidated: the Sma/MAD homologs and the Ras/mitogen-activated protein kinase pathways (79). In alachlor-induced tumors, TGFß masking protein, MAD-1, N-ras, and "ras related gene" (U12187) were highly upregulated. TGFß masking protein is a heat- and acid-stable protein that is inactivated by dithiothreitol and functions to neutralize the activity of TGFß in a dose-dependent manner (49, 69). Therefore, these studies suggest that dysregulation of TGFß signaling pathways may participate in both the initiation and progression of alachlor-induced olfactory mucosal carcinogenesis.
The human gene "deleted in malignant brain tumors 1" (DMBT1) is a candidate tumor suppressor for brain, gastrointestinal, and lung cancer and is altered or deleted in tumors of these tissues (46, 47). DMBT1, and related proteins in other species (rat ebnerin, mouse crp-ductin, rabbit hensin), are proposed to function in epithelial differentiation, immune defense, and mucosal protection (46, 47, 65, 67). DMBT1 was recently described as a mucin-like molecule that exists as transcripts of varying sizes, resulting from alternative splicing patterns, in different tissues (47). Rat ebnerin exhibits a single putative transmembrane domain that may participate in translocation of the protein to the plasma membrane for secretion (41). Similarly, the stratified squamous epithelium of the human esophagus constitutively targets DMBT1 to the extracellular matrix (47). Ebnerin was not detected by immunohistochemistry in normal olfactory mucosa, and, in contrast to other tumors wherein DMBT1 expression is lost or rearranged, alachlor-induced olfactory mucosal tumors express ebnerin in the lumens of the glandular portions of tumors, as well as on the epithelial surfaces of the tumors (Fig. 4). Increased expression of ebnerin was also reported in a model of liver injury and regeneration (4). The mechanism by which ebnerin gene expression is upregulated in either of these paradigms has not been elucidated, nor have activating mutations in DMBT1-related genes been described; these areas certainly deserve attention in light of the present findings.
In summary, we undertook these studies to determine whether the histological progression in alachlor-induced olfactory mucosal carcinogenesis is accompanied by gene expression changes that might yield clues as to the molecular pathogenic mechanisms for the formation of these tumors. The initial progression from histologically normal olfactory mucosa to foci of abnormal mucosa was accompanied by upregulation of genes consistent with a mutagenic response (Gadd45, apurinic/apyramidinic endonuclease 1), perhaps as a consequence of oxidative DNA damage [upregulation of heme oxygenase is a sensitive marker of oxidative stress (e.g., 52)]. Induction of multiple tubulin-related genes is likely at least in part related to the respiratory metaplasia that we observed, i.e., development of ciliated epithelial cells. In addition, evidence for early alteration of growth regulation was suggested by upregulation of genes associated with collagen degradation to facilitate cell growth (MMP-2 and MMP-9). It is likely that other genes within this cluster, including poorly understood or as yet undiscovered genes, represent additional modifiers of oncogenic initiation events. Their further characterization should allow us to refine our mechanistic understanding of the role of extracellular matrix remodeling in oncogenic processes (for review, see Ref. 48) and suggest potential therapeutic interventions (e.g., compounds described in Ref. 28). For example, inhibition of extracellular matrix remodeling enzymes might represent an effective mechanism to prevent tumor formation under some toxic exposure circumstances. Support for this hypothesis is provided by the previously mentioned phenotype of matrilysin-deficient mice, which is associated with decreased incidence of intestinal tumors in mice (1, 15, 78). Similarly, inhibition of MMP-2 binding to integrin by a small molecule has been shown to inhibit angiogenesis and tumor growth in vivo (61).
Progression from histologically altered olfactory mucosa to the development of adenomas was accompanied by expression of genes indicating inhibition of apoptosis [Bid3 (AI102299)] and enhancement of cell proliferation [e.g., zyxin, a focal-adhesion-associated phosphoprotein whose expression is directly related to cell spreading and proliferation and inversely related to differentiation (74)]. Progression to a malignant adenocarcinoma phenotype was characterized by induction of fibronectin, mucin 1, axin2, frizzled, as well as further downregulation of genes characteristic of the olfactory neuroepithelium, including sodium channel subunits, axonal-associated adhesion molecule, carboxylesterase 1, rhodanese, and cyclic nucleotide gated channel 4. The principal pathway that we have implicated in the late stage transformation to adenocarcinoma, the activation of nuclear ß-catenin, is also representative a pathway critical for oncogenesis in other organ systems and is associated with several well-characterized mutations in the wnt signaling pathway. Thus the alachlor-mediated oncogenesis model may be useful for addressing a number of therapeutic targets relevant to human cancer. The breadth of the genes and pathways that we have implicated provides a strong rationale for more detailed follow-up expanding to the entire genome and collecting additional biological replicates at each stage to sharpen the cluster-based categorizations of gene functions.
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ACKNOWLEDGMENTS
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We thank Sara Rankin for technical excellence, and Drs. Timothy P. Dalton and Kevin J. Mills for helpful discussions.
This publication was made possible by National Institute of Environmental Health Sciences (NIEHS) Grant ES-08799 (to M. B. Genter). The contents of this work are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS. B. J. Aronow acknowledges support from the Howard Hughes Medical Institute for the development of expression technology and bioinformatics infrastructure for the University of Cincinnati College of Medicine. Additional support was provided by NIEHS Grant P30-ES-06096, the University of Cincinnati Center for Environmental Genetics (M. W. Anderson, Principal Investigator).
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FOOTNOTES
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Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).
Address for reprint requests and other correspondence: M. B. Genter, Dept. of Environmental Health, ML 670056, Univ. of Cincinnati, Cincinnati, OH 45267-0056 (MaryBeth.Genter{at}UC.edu).
10.1152/physiolgenomics.00120.2002.
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