Reduction of Alachlor-Induced Olfactory Mucosal Neoplasms by the Matrix Metalloproteinase Inhibitor Ro 28-2653

Abstract

Chronic exposure to the chloracetanilide herbicide alachlor has been shown to cause olfactory mucosal neoplasms. Genomic analysis of olfactory mucosa from rats given alachlor (126 mg/kg/d) for from 1 day to 18 mo suggested that matrix metalloproteinases MMP-2 and MMP-9 were upregulated in the month following initiation of treatment. The present studies were designed to confirm this latter finding and to explore the potential role of MMPs in alachlor-induced olfactory carcinogenesis. Zymographic analysis of olfactory mucosal extracts confirmed that MMP-2 activity is higher in the olfactory mucosa of alachlor-treated rats. Therefore, rats were fed alachlor (126 mg/kg/d in the diet for 1 year) either with or without the MMP-2/MMP-9 inhibitor Ro 28-2653 (100 mg/kg daily by gavage for the first 2 months of alachlor treatment). The number of olfactory mucosal neoplasms was reduced by 25% after 1 year of alachlor treatment in rats that received both alachlor and Ro 28-2653. The morphology of alachlor-induced olfactory tumors was similar whether or not Ro 28-2653 had been given; the MMP inhibitor itself had no impact on olfactory mucosal histology. These data confirm that olfactory mucosal MMP-2 activity is increased following short-term alachlor exposure and show that administration of an MMP-2/9 inhibitor reduced the incidence of olfactory neoplasms in alachlor-treated rats, thereby implicating MMP-2 activity as a mediator of alachlor-induced carcinogenicity.

Introduction

Alachlor (2-chloro-2′,6′-diethyl-N-(methoxymethyl)acetanilide, CAS 15972-60-8) is one of several chloracetanilide herbicides in use worldwide in the production of crops such as corn, soybeans, peanuts, and rice. The chloracetanilides are multisite carcinogens in rodents, with olfactory mucosa, liver, stomach, and thyroid comprising the target sites for neoplasia in rats (U.S. EPA 1985, 1998). The mechanism of alachlor-induced olfactory mucosal mutagenicity (Wetmore et al., 1999) and carcinogenicity is proposed to involve metabolism via several intermediate steps, which can be catalyzed in part by human cytochrome P450 (CYP) 3A4, to 2,6-diethylaniline (DEA; Galati et al., 1998; Coleman et al., 1999). Subsequently, DEA is converted to a quinoneimine metabolite (Feng et al., 1990), and quinoneimines are known to deplete cellular antioxidants (Tee et al., 1987). Antioxidant depletion has been associated with oxidative DNA damage in other experimental systems (e.g., Guyton and Kensler, 1993; Feig et al., 1994; Ahmed et al., 1999; Iwai et al., 2000) and thus represents a possible mechanism of alachlor-induced mutagenicity and carcinogenicity.

An alternative mutagenic mechanism is transformation of DEA to a nitrosobenzene derivative (Kimmel et al., 1986) possibly by N-hydroxylation followed by acetylation (Guengerich, 1992). Acetylation activity in olfactory mucosa has been shown to significantly exceed that of liver using 4 different substrates (Genter, 2004). Recent work suggests that CYP2A3 is involved in the bioactivation of DEA, as the distribution of this enzyme in olfactory tissues from control rats correlates highly with the ultimate pattern of tumor formation in alachlor-treated animals (Genter et al., 2000).

Tumor induction and progression are complex processes, and perturbations in several other biochemical pathways involved in carcinogenesis have been attributed to alachlor. For example, alachlor-induced olfactory tumors that exhibit histological features consistent with aggressive clinical disease also have increased cytoplasmic accumulation and nuclear localization of β-catenin (Genter et al., 2002b), accumulation of which accompanies activation of the Wnt signaling pathway. Wnt signaling is implicated in neoplastic initiation and progression in many tissues (Moon et al., 2004). Similarly, a recent genomic study suggested that expression of oxidative stress and matrix metalloproteinase (MMP) genes (specifically MMP-2 and MMP-9) is upregulated in the olfactory mucosa of rats in the first month following initial alachlor exposure. No other nongelatinase MMPs were regulated by alachlor exposure (Genter et al., 2002b).

We undertook the present studies in order to determine whether the upregulation of MMP-2 and MMP-9 gene expression in the olfactory mucosa of alachlor-treated rats was associated with enhanced activity, and to examine the impact of the MMP-2 and-9 inhibitor Ro 28-2653 on alachlor-induced tumor formation. Ro 28-2653 is orally bioavailable and is highly selective toward MMP-2, MMP-9, and membrane type 1-MMP (Grams et al., 2001). Ro 28-2653 treatment has previously been demonstrated to increase apoptosis and survival in a rat model of prostate cancer (Lein et al., 2002) and to reduce tumorigenesis, tumor invasiveness, and angiogenesis in a battery of in vitro and in vivo assays (Maquoi et al., 2004).

Materials and Methods

Reagents

Alachlor (>98% pure) was obtained from Chem Service (West Chester, PA). Ro 28-2653 was acquired from Roche Diagnostics (Penzberg, Germany). Electrophoresis supplies were obtained from Biorad (Hercules, CA). Brij-35 was obtained from Fisher Scientific (Fair Lawn NJ). All other materials were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted.

Animals

This work was performed using the same rat model of alachlor-induced olfactory mucosal neoplasia that we have described previously, namely feeding alachlor at 126 mg/kg/d mixed into powdered diet (Genter et al., 2000, 2002a, 2002b). Experiments were preapproved by the University of Cincinnati Institutional Animal Care and Use Committee.

For all studies, male Long-Evans rats (4–5 weeks of age; Harlan Indianapolis, IN) were acclimatized for 1 week before being randomly assigned to treatment groups. Animals had free access to tap water and were fed a powdered rodent chow (Harlan). All rats were maintained under standard environmental conditions (12 hours light; temperature, 22 ± 1°C; relative humidity, 50 ± 10%).

Treatments

Olfactory MMP Activity Study by Zymography

Rats (n = 4 per group) were fed 0 (no alachlor in diet) or 126 mg/kg/day (mixed uniformly in diet) for 10 days. Rats were sacrificed by carbon dioxide asphyxiation, and the olfactory mucosa from the nasal septum was placed in 500 uL of cold zymography buffer (recipe below; on ice) until homogenization (<1 hour). Ethmoid turbinates were snap frozen on dry ice and stored at −80 for possible future analyses.

Activities of MMP-2 and MMP-9 were semi-quantified by zymographhy as described (Porter et al., 1999), with minor modifications. The septal olfactory mucosa was homogenized (2 mM urea, 50 mM Tris, pH 7.6, 137 mM NaCl, 1 g/L EDTA, 0.1% Brij-35, 0.1 mM phenylmethanesulfonyl fluoride [PMSF]) and dialyzed (12–14,000 kDa cutoff) against dialyzing buffer (25 mM Tris, pH 8.5, containing 10 mM CaCl₂, 0.1% Brij-35, 0.1 mM PMSF) for 12–18 hours at 4°C. Protein concentrations were determined using the Biorad (Hercules, CA) protein reagent. A 20 μg aliquot of each sample was separated by electrophoresis under nondenaturing conditions (i.e., no reducing agent was used) through a 10% polyacrylamide gel containing gelatin (0.1%) using prestained molecular weight markers (Biorad). After separating the proteins, SDS was removed from the gel by washing in 4 changes of 2.5% Triton X-100 over the course of 1 hour. The gel then was immersed for 18 hours in incubation buffer (50 mM Tris, pH 7.4 containing 10 mM CaCl₂, 0.05% Brij-35) at 37°C with shaking. Gels were fixed and stained (0.1% Coomassie blue, 50% methanol, 20% acetic acid) for 2–3 hours and rinsed several times with deionized water. Distinct bands for MMP-9 (~92 kD), pro-MMP-2 (~72 kD), and active (~66 kD) were evident as clear bands against a blue background (Porter et al., 1999).

In Vivo MMP Inhibition Study

Rats received 1 of 5 treatments (Table 1): none; vehicle (0.2% carboxymethyl-cellulose [CMC] 5 ml/kg/d body weight (b.w.) by gavage); Ro 28-2653 alone (100 mg/kg/d in CMC by gavage at 5 ml/kg b.w.); alachlor alone (126 mg/kg/d in powdered diet for 12 months); or alachlor (126 mg/kg/d in the diet) + Ro 28-2653 (100 mg/kg/d in CMC by gavage at 5 ml/kg total body weight). All animals treated by gavage were lightly anesthetized for ~2 minutes with isoflurane prior to gavage treatment. For both groups receiving Ro 28-2653, the inhibitor was given daily for 2 months based on our prior results that suggested that alachlor-induced MMP upregulation occurs during the first month of alachlor exposure (Genter et al., 2002b). A subset of rats from each group was weighed weekly so that the alachlor concentration in the diet and/or volume of CMC could be adjusted to maintain the target dose in the growing rats. Two animals given Ro 28-2653 alone as well as 2 rats treated with CMC only were necropsied immediately after completion of the 2-month treatment regimen to determine whether or not the vehicle or inhibitor induced structural changes in the olfactory mucosa. The remaining rats were retained for an additional 10 months, receiving either no treatment (groups 1 and 3) or alachlor at 126 mg/kg/d (groups 4 and 5).

At necropsy, animals were asphyxiated with carbon dioxide. Nasal cavities, tracheas at the level of the thyroid gland, and liver samples were prepared for histology. To prepare nasal cavities for histology, the lower jaw was removed from the head and the nasal cavity flushed via the nasopharynx with 5 ml of neutral buffered 10% formalin. The head was further fixed by immersion in fresh formalin for an additional 24 to 48 hours and then transferred into 10 L of 10% formic acid containing 500 g of Rexyn ion exchange resin for 10 days of decalcification with stirring. Decalcified tissues were washed overnight in cold tap water, trimmed transversely at 4 levels (Young, 1981), and then all tissues were embedded in paraffin using standard methods. Serial 5-μm-thick sections were stained with hematoxylin and eosin (H&E) or by immunohistochemistry to detect β-catenin (using a monoclonal β-catenin primary antibody [1:100 dilution; BD-Gentest] and TSA enhancement [Perkin Elmer] as previously described [Genter et al., 2002b]). All tissue sections were examined by light microscopy. Neoplastic lesions were counted at all 4 levels of the nasal cavity in both H&E and β-catenin-stained sections to determine the number of neoplasms per rat. Immunostained sections also were used to localize β-catenin expression in each mass.

Statistical Analysis

All results were expressed as the mean ± standard error. Data were compared using a 2-tailed t-test test. A p-value of 0.05 was used to delineate significant differences between groups.

Results

MMP Activities

* Zymographic analysis revealed an increase in fully active MMP-2 in the olfactory mucosa of rats that had been fed alachlor at a carcinogenic level (126 mg/kg/day) for 10 days (Figure 1). Increased MMP-9 activity was not detected in alachlor-treated rat olfactory mucosa.

Effect of Ro 28-2653 on Alachlor-Induced Tumor Formation

In vivo inhibition of MMP activity by administration of Ro 28-2653 significantly (p = 0.05) reduced the incidence of olfactory mucosal neoplasms in rats fed a carcinogenic dose of alachlor (126 mg/kg/day) for 1 year. The animals that received both Ro 28-2653 and alachlor had a mean tumor number of 3.6 ± 0.3 (mean ± SEM) as compared to a mean tumor number of 4.8 ± 0.3 in the rats receiving alachlor only.

The histological features in alachlor treated and alachlor + Ro 28-2653-treated rats was very similar, and is summarized in Figure 2. As described previously, the most common olfactory mucosal neoplasm induced by alachlor was the well-differentiated polypoid adenoma, typically presenting as a space-occupying mass with narrow stalk, comprised of ciliated pseudostratified epithelium covering a fine fibrovascular core, and with few mitotic figures (Genter et al., 2000, 2002a). Treatment with Ro 28-2653 did not alter the morphologic phenotype of alachlor-induced masses, nor did administration of the inhibitor elicit tumors with novel histologic features. β-Catenin immunoreactivity was present in olfactory tumors from 3 rats treated with both alachlor and Ro 28-2653 (Figure 3). No β-catenin was noted in neoplasms of rats exposed to alachlor alone, or in the olfactory mucosa of any of the other treatment groups.

Extended treatment with Ro 28-2653 was well tolerated. Rats given Ro 28-2653 at 100 mg/kg/d for 2 months displayed a modest reduction in body weight gain compared to untreated controls, but the rate of body weight gain normalized upon cessation of inhibitor treatment. Furthermore, animals treated with Ro 28-2653 exhibited no signs of systemic toxicity either during the 2-month treatment regimen or the 10-month holding period. The structures of olfactory mucosa, thyroid, and liver from rats given the MMP inhibitor but not exposed to alachlor were indistinguishable from those of untreated animals both at the end of the 2-month Ro 28-2653 phase and at the conclusion of the 10-month holding (data not shown).

Discussion

Recent results from our laboratory suggest that dysregulation of extracellular matrix genes and antioxidant perturbations may act together in the pathogenesis of alachlor-induced olfactory tumors. Biochemical studies from our laboratory confirm that alachlor and metolachlor induce antioxidant perturbations in target tissues for carcinogenicity induced by the respective compounds (Burman et al., 2003; also unpublished observations from Genter lab). Gene expression studies suggested that MMP-2 and MMP-9 were upregulated following short-term alachlor exposure, concomitant with upregulation of oxidative stress-related genes (Genter et al., 2002b). Therefore, we have proposed that MMP upregulation is involved in the initiation, rather than the progression of alachlor-induced olfactory tumors. The present study (Figure 1) confirms that MMP-2 activity is enhanced in the olfactory mucosa of alachlor-treated rats, but not MMP-9. Microarray studies are often associated with “false positive” responses—i.e., indicating changes in gene expression that cannot be confirmed by other means. This observation highlights the need for independent verification of microarray gene expression results in advance of attributing biological significance to these observations.

Recent studies from other laboratories have demonstrated an association between oxidative stress, antioxidant levels, and expression of MMPs. MMP-9 levels were elevated in bronchoalveolar lavage fluid from neonates with respiratory distress, which was associated with oxidative damage (Schock et al., 2001). In a bovine aortic endothelial cell (BAEC) model of diabetes mellitus, exposure of BAEC to glucose resulted in increased reactive oxygen species generation and increase in MMP-9 activity and expression; reduction of reactive oxygen species with antioxidants decreased glucose-induced MMP-9 activation (Uemura et al., 2001). Both MMP-2 and MMP-9 were upregulated upon induction of oxidative stress in cardiac fibroblasts, an effect that was reversed upon administration of a superoxide dismutase/catalase mimetic (Siwik et al., 2001).

The present study showed that a 2-month administration of the MMP inhibitor Ro 28-2653 significantly reduced the number of olfactory tumors per rat in alachlor-treated rats. Histologically, the tumors in both alachlor-treated groups appeared quite similar. There was a range of size of tumors, ranging from very small nodules to large tumors filling a portion of the nasal airways in both alachlor treatment groups. The inhibitor itself did not cause any microscopic alterations of the olfactory mucosa, and the animals tolerated the treatment well. In our previous studies, we found that β-catenin expression was correlated with progression of alachlor-induced neoplasms to a more aggressive phenotype and was generally found only in advanced lesions (e.g., those occupying a significant fraction of the rats’ nasal airways). In contrast, in the present study, β-catenin was only detected by immunohistochemistry in rats treated with alachlor + Ro 28-2653 (i.e., not in neoplasms in rats treated with alachlor alone), and was found in smaller neoplasms than in our previous study. The biological basis for this observation is not obvious but might indicate that Ro 28-2653 treatment retards the initial growth of tumors but not the biological progression.

There is also significant evidence of an important role of MMP regulation in cancer, as MMPs are proposed to be involved in promoting cancer cell invasiveness. Upregulation/activation of MMP-2 and MMP-9 are associated with tumor progression and poor prognosis in human breast cancers, with MMP-9 immunoreactivity highly associated with infiltrating lobular carcinomas (Jones et al., 1998). Increased expression of MMP-2 and MMP-9, as well as “tissue inhibitor of metalloproteinase” (TIMP)-1 and TIMP-2 correlated with poor prognosis in human renal cell carcinomas (Kallakury et al., 2001). MMP-9 was also associated with invasive murine breast carcinomas (Kupferman et al., 2000). MMP-2 overexpression was associated with transformation of lens epithelial cells in vitro (Seomun et al., 2001). In addition, MMP-2 was identified as an overexpressed gene in human head and neck cancers (Villaret et al., 2000).

Inhibition of MMPs appears to have a therapeutic effect in other animal tumor models. Matrilysin (MMP-7) is important in the early stages of intestinal tumorigenesis, and ablation of MMP-7 significantly reduced tumor formation in 2 models of intestinal tumorigenesis (Wilson et al., 1997; Goss et al., 1998). All-trans retinoic acid, 9-cis retinoic acid, and 13-cis retinoic acid reduced MMP-7 expression and suppressed the invasiveness of colon cancer cell lines in vitro and in mice in vivo (Adachi et al., 2001). The broad-spectrum MMP inhibitor batimastat greatly decreased the incidence of intestinal tumors in mice heterozygous for the adenomatous polyposis coli (Apc) tumor suppressor gene (Goss et al., 1998). Three different MMP-7 inhibitors displayed efficacy in increasing survival time and/or decreasing tumor number and aggressiveness in rodent models (Wilson et al., 1997; Goss et al., 1998; Adachi et al., 2001), making MMP genes potential targets for abrogating alachlor-induced olfactory mucosal tumors.

The results presented herein represent a “proof of concept” that MMP inhibition may represent a means to reduce or eliminate the olfactory carcinogenicity of chloracetanilide herbicides. In the present studies, Ro 28-2653 (or vehicle alone) was administered daily to rats for 2 months, and a 25% reduction in average number of tumors per rat was observed with Ro 28-2653 treatment. It is difficult to speculate as to whether longer treatment would afford more protection, because up-regulation of MMP-2 gene expression was seen in the first month of alachlor treatment (Genter et al., 2002b), and in the present study we dosed with the inhibitor for 1 month longer than that. Further, an even more extensive inhibition regimen (8 weeks of daily batimastat administration to Apc^Min/− mice, which are genetically predisposed to intestinal tumors) did not completely abolish tumor formation (Goss et al., 1998). The present studies were labor intensive (given that daily anesthesia and gavage dosing were involved), so we propose that future (longer-term) studies be conducted with a MMP-2 inhibitor of sufficient water solubility and palatability such that the animals could be treated via the drinking water, thus reducing technical effort and animal handling and stress.