ToxSci Advance Access originally published online on November 8, 2006
Toxicological Sciences 2007 95(2):331-339; doi:10.1093/toxsci/kfl158
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Published by Oxford University Press 2006.
Inhibition of Urethane-Induced Carcinogenicity in Cyp2e1–/– in Comparison to Cyp2e1+/+ Mice
Laboratory of Pharmacology and Chemistry, National Toxicology Program, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709
1 For correspondence via fax: (919) 541-4632. E-mail: ghanayem{at}niehs.nih.gov.
Received September 12, 2006; accepted November 3, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Urethane is an established animal carcinogen and has been classified as "reasonably anticipated to be a human carcinogen." Until recently, urethane metabolism via esterase was considered the main metabolic pathway of this chemical. However, recent studies in this laboratory showed that CYP2E1, and not esterase, is the primary enzyme responsible for urethane oxidation. Subsequent studies demonstrated significant inhibition of urethane-induced genotoxicity and cell proliferation in Cyp2e1–/– compared to Cyp2e1+/+ mice. Using Cyp2e1–/– mice, current studies were undertaken to assess the relationships between urethane metabolism and carcinogenicity. Urethane was administered via gavage at 1, 10, or 100 mg/kg/day, 5 days/week, for 6 weeks. Animals were kept without chemical administration for 7 months after which they were euthanized, and urethane carcinogenicity was assessed. Microscopic examination showed a significant reduction in the incidences of liver hemangiomas and hemangiosarcomas in Cyp2e1–/– compared to Cyp2e+/+ mice. Lung nodules increased in a dose-dependent manner and were less prevalent in Cyp2e1–/– compared to Cyp2e+/+ mice. Microscopic alterations included bronchoalveolar adenomas, and in one Cyp2e1+/+ mouse treated with 100 mg/kg urethane, a bronchoalveolar carcinoma was diagnosed. Significant reduction in the incidence of adenomas and the number of adenomas/lung were observed in Cyp2e1–/– compared to Cyp2e1+/+ mice. In the Harderian gland, the incidences of hyperplasia and adenomas were significantly lower in Cyp2e1–/– compared to Cyp2e+/+ mice at the 10 mg/kg dose, with no significant differences observed at the high or low doses. In conclusion, this work demonstrated a significant reduction of urethane-induced carcinogenicity in Cyp2e1–/– compared to Cyp2e1+/+ mice and proved that CYP2E1-mediated oxidation plays an essential role in urethane-induced carcinogenicity.
Key Words: Cyp2e1–/– mice; urethane metabolism; urethane carcinogenicity; lung tumors; liver tumors; Harderian gland tumors.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Epoxides are known potent mutagens and carcinogens. A great number of chemicals are metabolized via cytochromes P450 (CYPs) leading to the formation of epoxide intermediates. In particular, CYP2E1 is implicated in the bioactivation of a wide range of procarcinogens via epoxide formation, including benzene, vinyl chloride, 1,1-dichloroethylene, trichloroethylene, 1,3-butadiene, acrylonitrile, and acrylamide.
With the identification of hundreds of CYP proteins and the cloning and sequencing of their cDNA's in recent years, it became possible to produce CYP knock out mice that lack the ability to express specific isozymes, including CYP2E1 (Lee et al., 1996
). These animals presented valuable models for the reinvestigation of the metabolism, toxicity, genotoxicity, and carcinogenicity of many chemicals and drugs. Using urethane (ethyl carbamate, EC) as a model chemical, the current investigations were undertaken as part of an overall project intended to assess the role of epoxidation in the genotoxicity and carcinogenicity of epoxide-forming chemicals using Cyp2e1–/– mice. Urethane was selected because it is a well-studied chemical, an established potent animal carcinogen that has a short onset for tumor development in mice (Inai et al., 1991; Kristiansen et al., 1990; Nettleship et al., 1943; Schmahl et al., 1977) and is present in a variety of fermented foods and beverages consumed by humans (Salmon and Zeise, 1991). Urethane is formed as a by-product of fermentation in a variety of foods and beverages (including bread, cheese, fruit bandies, wine, and beer) and is also found in tobacco leaves and smoke (Salmon and Zeise, 1991).
Most recent studies have confirmed that urethane is a multisite carcinogen in both male and female B6C3F1 mice (NTP, 2004). Nomura (1975) demonstrated in a multigenerational study that a single subcutaneous injection of urethane (1.0 mg/g body weight) on gestation day 17 caused tumor formation at multiple sites in first generation (F1) offspring. Furthermore, mating among F1 mice produced a second generation exhibiting the same pattern of tumor development (Nomura, 1975). Urethane is also a potent mutagen (Schmahl et al., 1977; Witt et al., 2000). In Salmonella typhimurium, in the presence of liver S9, urethane induces sister chromatid exchanges, induces sex-linked recessive lethal mutations, and reciprocal translocations in germ cells of male Drosophila melanogaster (Zeiger et al., 1992). Urethane also caused a significant increase in the frequency of micronucleated erythrocytes in peripheral blood and in bone marrow of male and female B6C3F1 mice (Hoffler et al., 2005; Witt et al., 2000).
It has been accepted that urethane metabolism (Fig. 1) occurs via two major pathways (Forkert and Lee, 1997; Hoffler et al., 2003; Lee et al., 1998; Yamamoto et al., 1990). The first pathway of urethane metabolism was thought to be catalyzed by esterase leading to the formation of CO2, ethanol, and NH3. Studies conducted in rats and mice (Hoffler et al., 2003; Mirvish, 1968; Nomeir et al., 1989; Salmon and Zeise, 1991; Skipper et al., 1951; Yamamoto et al., 1988) using 14C-urethane demonstrated that greater than 90% of the administered dose was exhaled as 14CO2 within 24 h. The second pathway is thought to involve oxidative metabolism of urethane via CYP enzymes and was thought of as a bioactivation pathway. It was proposed that CYPs were responsible for urethane's bioactivation via vinyl carbamate (VC) formation and subsequent metabolism of VC to vinyl carbamate epoxide (VCE), was suggested to occur via CYPs as well (Dahl et al., 1978, 1980; Hoffler et al., 2003, 2005). VCE is highly reactive with DNA and is considered the ultimate carcinogenic metabolite of urethane (Dahl et al., 1978, 1980; Guengerich and Kim, 1991; Park et al., 1990). Studies by Guengerich and Kim (1991) indirectly showed that CYP2E1 is involved in the epoxidation of urethane. Human liver microsomes were incubated with either urethane or VC in the presence of adenosine and a NADPH-generating system. 1,N6-ethenoadenosine adducts were detected as a result of exposure to these chemicals. In subsequent in vitro incubations that included the CYP2E1 inhibitor, diethyldithiocarbamate, or CYP2E1 antibodies, inhibition of adduct formation was observed. Moreover, 1,N6-ethenoadenosine and 3,N4-ethenocytidine adducts were also detected in hepatic RNA after a single injection of urethane to 12-day-old and adult male mice (Ribovich et al., 1982).
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Until recently, the prevailing hypothesis stated that while esterase is the primary enzyme responsible for urethane metabolism, CYP2E1 is a minor pathway leading to urethane oxidation (Forkert and Lee, 1997
; Lee et al., 1998; Nomeir et al., 1989; Yamamoto et al., 1990). Unfortunately, most of the earlier evidence relating to the enzymatic basis of urethane metabolism was indirect and frequently relied on the use of nonspecific metabolism modulators. Most recently, however, studies by Hoffler and Ghanayem (2005) and Hoffler et al. (2003, 2005) using Cyp2e1–/– null mice proved that contrary to earlier reports, CYP2E1, and not esterase, is the principal enzyme responsible for the metabolism of urethane. Subsequent studies in this laboratory demonstrated significant inhibition of urethane-induced genotoxicity and cell proliferation in Cyp2e1–/– versus Cyp2e1+/+ mice (Hoffler et al., 2005). Present work was undertaken to assess the role of CYP2E1-mediated oxidation in the carcinogenicity of urethane using Cyp2e1–/– and Cyp2e1+/+ mice. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals.
EC (urethane), with a stated purity of > 98%, was obtained from TCI America (Tokyo, Japan). All other chemicals were of the best commercially available purity.
Animals and treatment.
Cyp2e1–/– (CYP2E1 null) and Cyp2e1+/+ (wild type) mice were obtained from the National Cancer Institute, Bethesda, MD. The development and background of these mice were as previously reported (Hoffler et al., 2003; Lee et al., 1996). Subsequently, animals were rederived and bred at Charles River Laboratories (Wilmington, MA). Western Blot analysis was used to confirm the nullizygosity of Cyp2e1–/– mice as previously described (Wang et al., 2002).
In this work, 4- to 5-weeks old male Cyp2e1+/+ and Cyp2e1–/– mice ranging in weight from 20–24 g were used. Animals were randomized based on body weight, divided into groups (28–30 animals each), and individually housed in facilities with a 12-h light-dark cycle, and National Institutes of Health (NIH) #31 diet and water were available ad libitum throughout the experiments. Mice were kept in our animal facilities for 1 week to acclimate prior to chemical administration. All animal care and experimentation were conducted according to NIH guidelines (U.S. Department of Health and Human Services, 1985).
Urethane dosing solutions were daily prepared in tap water in a dose volume of 10 ml/kg and administered by gavage at 1, 10, or 100 mg/kg/day, 5 days/week, for 6 weeks. Matching controls received 10 ml water/kg/day by gavage.
Twenty-four hours after the last urethane dose, 14–15 animals from each dose group were euthanized using CO2/O2. Tissues were harvested and placed in 10% neutral buffered formalin for 18–20 h and then transferred to 70% ethanol fixative.
The remaining animals (14–15 animals/group) were allowed to recover without chemical administration for 7 months. At the end of the 7-month recovery period, animals were euthanized using CO2/O2, and tissues were collected and processed as described above.
Tissues from all animals were routinely processed, and H & E–stained sections were prepared for histopathological evaluation as described above.
Histopathological evaluation.
A complete gross necropsy and microscopic examination were performed on all mice. At necropsy, all organs and tissues were examined for grossly visible lesions, removed, and fixed in 10% neutral buffered formalin for 18–20 h and then transferred to ethanol. The lungs were infused with 10% formalin, removed, and placed in formalin for 18–20 h and then transferred to 70% ethanol. Tissues were trimmed, embedded in paraffin, sectioned to a thickness of 4–6 µm, and stained with hematoxylin and eosin for microscopic examination. For all paired organs (e.g., adrenal gland and kidney), samples from each organ were examined. Representative sections of the following organs were examined: liver, gall bladder, lungs, adrenal glands, femur, brain, spinal cord, gall bladder, Harderian glands, heart with aorta, kidney, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), lymph nodes (mandibular and mesenteric), nasal cavity, pancreas, preputial gland, prostate, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, esophagus, urinary bladder, and all gross lesions. All animals that died prior to the scheduled sacrifice were immediately opened and placed in formalin and processed as described above. Tissues from these animals that were judged to be acceptable for processing and microscopic evaluation were also processed to slides, examined, and included in the final tables and analysis. An experienced board certified pathologist first evaluated the tissues microscopically, and the findings were tabulated. Subsequently, a second pathologist evaluated all tissues with lesions and the diagnosis was confirmed or corrected. The two pathologists then conferred with a third pathologist and evaluated and discussed all discrepancies and agreed on a final diagnosis.
Statistical analysis.
Analysis of the incidences of lung, Harderian gland, and liver lesions across dose groups were compared using the Cochran-Armitage trend test, and the p values reported for these comparisons are one sided.
The incidences of lesions between the treated and control groups and between the incidences in Cyp2e1+/+ compared to Cyp2e1–/– mice were compared using Fisher exact test. The p values reported for these comparisons are one sided.
Comparison of the number of tumors per lung across doses within each genotype of mice and also the number of tumors per lung between the two genotypes for each dose group were performed using ANOVA, and the reported p values are two sided.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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No obvious signs of toxicity were noted during the 6 weeks of dosing (data not shown). However, at necropsy 24 h after the last administered urethane dose, keratosis or keratinization of the nonglandular forestomach was noted in 3 of 9 Cyp2e1+/+ and 5 of 10 Cyp2e1–/– mice dosed with 100 mg urethane/kg/day for 6 weeks. This alteration was considered to be urethane related, essentially reversible, since the only macroscopic evidence of previous gastric injury noted later on at the end of the recovery period was a solitary nodule; this was histologically confirmed to be epithelial hyperplasia and hyperkeratosis (noted in 1 of 15 Cyp2e1+/+ mice dosed with 100 mg/kg urethane).
No other significant macroscopic alterations or histopathological lesions that can be attributed to urethane were observed at the end of the 6-week dosing regimen in mice of either genotype.
Chemical-Induced Microscopic Lesions in Cyp2e1–/– and Cyp2e1+/+ Mice
At the end of the 7 months recovery period after urethane administration for 6 weeks, chemical-related macroscopic alterations were noted in the lungs, livers, and Harderian gland of urethane-dosed mice; however, these alterations were more prevalent in Cyp2e1+/+ than Cyp2e1–/– mice. The liver and lung alterations included foci and nodules, respectively, with increasing frequency and multiplicity as the urethane dose increased and with the lesions more pronounced in Cyp2e1+/+ than in Cyp2e1–/– mice.
Urethane-induced histopathologic alterations in the liver appeared to be strongly dependent on CYP2E1 including hepatocellular hypertrophy, eosinophilic foci, angiectasis, and vascular tumors (hemangiomas and hemangiosarcomas) as evident from the higher incidence of lesions in Cyp2e1+/+ compared to Cyp2e1–/– mice (Table 1). Hepatocellular hypertrophy was present in 0, 1, 4, and 12 Cyp2e1+/+ mice but was noted in only 1, 0, 0, and 1 Cyp2e1–/– mice at 0, 1, 10, and 100 mg/kg, respectively. The incidence of hepatocellular hypertrophy was significantly higher in Cyp2e1+/+ compared to Cyp2e1–/– at both the 10 and 100 mg urethane/kg (Table 1). Eosinophilic foci were present in 0, 0, 1, and 9 Cyp2e1+/+ mice and observed in only one vehicle-treated Cyp2e1–/– mice. Angiectasis was observed in 0, 0, 3, and 11 Cyp2e1+/+ mice and 0, 0, 0, and 2 Cyp2e1–/– mice at 0, 1, 10, and 100 mg/kg, respectively (Table 1). Angiectasis was differentiated from hemangioma by the relative absence of cellularity and architectural disruption. The increase in the incidence of the eosinophilic foci and angiectasis in the high urethane dose Cyp2e1+/+ mice was statistically significant in comparison to vehicle-treated Cyp2e1+/+ mice and high-dose Cyp2e1–/– mice (Table 1). Liver vascular tumors (hemangioma and hemangiosarcoma) were observed primarily in Cyp2e1+/+ mice treated with the high urethane dose, and the incidence of hemangiomas (5/15) was significantly higher than in the corresponding vehicle-treated controls. One Cyp2e1–/– mouse treated with the high dose of urethane exhibited a hemangioma (Table 1). Hemangiosarcomas were also observed in the high dose Cyp2e1+/+ mice, and the incidence (8/15) was statistically significant in comparison to vehicle-treated Cyp2e1+/+ mice as well as high dose Cyp2e1–/– mice (Table 1).
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Lung macroscopic observations were clearly less frequent in Cyp2e1–/– compared to Cyp2e1+/+ mice, and the multiplicity (number of lesions per lung of affected mice) of lung alterations was also reduced in Cyp2e1–/– compared to Cyp2e1+/+ mice (Fig. 2, Table 2). Most of the lung alterations correlated histologically with bronchoalveolar tumors. The spectrum of lung microscopic alterations noted in this study included bronchoalveolar epithelial hyperplasia, characterized by epithelial proliferation with preservation of overall pulmonary architecture. With all lesions, the incidences of urethane-induced lesions were higher in Cyp2e1+/+ compared to Cyp2e1–/– mice (Table 2). Although there were no hyperplastic changes observed in the lungs of control or low-dose mice of either genotype, the incidences of bronchoalveolar hyperplasia were increased at both the middle and high doses of urethane in comparison to vehicle-treated controls in mice of both genotypes (Table 2). The incidence of bronchoalveolar hyperplasia was higher in Cyp2e1+/+ mice; however, it was not statistically different when compared to the incidence in Cyp2e1–/– mice (Table 2). Bronchoalveolar adenomas in mice were characterized by expansive epithelial proliferative alterations causing compression of adjacent lung tissue. In one Cyp2e1+/+ mouse treated with 100 mg/kg urethane, a less-differentiated bronchoalveolar carcinoma was observed (Table 1). The bronchoalveolar adenomas were dose related, tending to be more severe and multifocal or multiple with increased urethane dose and were at least partially dependent upon the CYP2E1-mediated metabolism of this chemical since the incidence of adenomas and the number of adenomas per lung significantly declined in Cyp2e1–/– compared to Cyp2e1+/+ mice at all urethane doses (Table 2).
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The frequency of Harderian gland hyperplasia and adenoma were also dose related and appeared to be, at least partially, dependent upon CYP2E1-mediated metabolism of urethane, being somewhat less frequent in Cyp2e1–/– than in Cyp2e1+/+ mice (Table 3). These lesions were dose dependent in mice of both genotypes and their incidence apparently reached maximum at the 10 mg/kg in Cyp2e1+/+ mice since it was roughly similar at both the medium and high doses (Table 3). Further, although the incidences of Harderian gland adenomas were higher in Cyp2e1+/+ compared to Cyp2e1–/– mice at all doses of urethane, the increase in the incidence of these tumors was statistically significant at the 10 mg/kg dose only (Table 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Urethane (EC) is a potent multisite carcinogen capable of inducing tumors in various organs and animal species regardless of the route of administration (Mirvish, 1968
; NTP, 2004; Salmon and Zeise, 1991) and has been classified as "reasonably anticipated to be a human carcinogen" (NTP, 2000). Consumption of fermented foods and alcoholic beverages containing urethane is considered the primary source of human exposure to this chemical. Due to its presence in alcoholic beverages, and the fact that ethanol is a substrate and inducer of CYPs which were implicated in metabolizing urethane, this chemical was recently tested alone or in combination with ethanol for carcinogenicity in mice (NTP, 2004). Results of this work demonstrated that administration of urethane alone in drinking water resulted in "clear evidence" of carcinogenicity in male B6C3F1 mice based on increased incidences of lung, liver, Harderian gland, skin, and forestomach tumors, and hemangiosarcoma in the liver and heart. In female B6C3F1 mice, these studies demonstrated "clear evidence" of carcinogenicity as demonstrated by an increase in the incidence of lung, liver, Harderian gland, mammary gland, and ovarian tumors and hemangiosarcoma in the liver and spleen (Beland et al., 2005; NTP, 2004). In these studies, coadministration of urethane with ethanol in drinking water produced weak and inconsistent evidence for an interaction between the two chemicals in the carcinogenicity of urethane.
Urethane metabolism has long been considered a prerequisite for the induction of genotoxicity and carcinogenicity by this chemical. Until recently, urethane metabolism (Fig. 1) via an esterase enzyme leading to the formation of CO2, NH3, and ethanol was considered the primary pathway in animals and exhaled CO2 was identified as the main metabolite of this chemical, accounting for 90–95% of dose (Hoffler et al., 2003; IARC, 1974; Nomeir et al., 1989; Salmon and Zeise, 1991). Because esterase-mediated metabolism leads primarily to CO2 formation, we considered this pathway a detoxification pathway and therefore could not account for the potent genotoxicity and carcinogenicity of urethane. A second minor pathway is N-hydroxylation leading to the formation of N-hydroxyurethane, which has also been reported (Mirvish, 1968) and accounted for less than 1.0% of urethane metabolism (Salmon and Zeise, 1991). Studies by Kaye and Trainin (1966) and Mirvish (1968) demonstrated that N-hydroxyurethane may undergo conversion to urethane, and it is a less potent carcinogen than the parent compound. Therefore, it became obvious that this pathway may not explain the potent carcinogenic activity of urethane.
A third pathway that was reported to account for less than 1.0% of urethane metabolism (Salmon and Zeise, 1991) involves the oxidation of urethane to form VC (Fig. 1) was also proposed (Dahl et al., 1978, 1980). More recently, Hoffler and Ghanayem (2005) proposed that urethane might also undergo C-hydroxylation leading to the formation of VC (Fig. 1). VC may undergo subsequent metabolism leading to the formation of VCE (Fig. 1). Thus, our primary focus centered on the epoxidation of urethane to VCE as the primary mechanism leading to the bioactivation of this chemical. Studies by Lawson and Pound (1973) indirectly supported this premise in showing that the ethyl carbons of urethane become covalently bound to mouse DNA in vivo. Further, Dahl et al. (1978, 1980) demonstrated that VC is 10–50 times more potent as a carcinogen than urethane in mice, and Park et al. (1993) showed that VC is a less potent carcinogen than VCE. Subsequent in vitro studies demonstrated that human CYP2E1 was involved in the oxidation of urethane (Guengerich and Kim, 1991). With the focus on oxidation via CYP enzymes as a necessary step leading to the genotoxicity and carcinogenicity of urethane, we felt that the reported contribution of CYP enzymes at less than 1.0% of urethane metabolism (Salmon and Zeise, 1991) may not be quantitatively sufficient to explain the potent genotoxicity and carcinogenicity of this chemical. We therefore hypothesized that CYP2E1-mediated oxidation, and not esterase-mediated hydrolysis, is the primary pathway responsible for urethane bioactivation via epoxidation (Fig. 1) and the subsequent genotoxicity and carcinogenicity. Accordingly, the overall objectives of the current studies were to assess the role of CYP2E1-mediated oxidation of urethane in the metabolism, genotoxicity, and carcinogenicity of this chemical using Cyp2e1–/– mice.
Using Cyp2e1–/– mice, the first series of studies in this laboratory demonstrated that while the contribution of CYP2E1 to urethane metabolism exceeds 96%, esterase contribution accounted for less than 1% (Hoffler and Ghanayem, 2005; Hoffler et al., 2003, 2005). The contribution of CYPs other than CYP2E1 accounted for approximately 3.5% of urethane metabolism. Subsequent studies in this laboratory demonstrated a significant reduction of urethane-induced genotoxicity and cell proliferation in the liver and lung of Cyp2e1–/– compared to Cyp2e1+/+ mice (Hoffler et al., 2005). Using Cyp2e1–/– and Cyp2e1+/+ mice, current studies were undertaken to address the hypothesis that in vivo bioactivation of urethane via CYP2E1 is a prerequisite for the induction of tumors by this chemical.
Urethane administration for 6 weeks (5 days/week) at doses comparatively similar to doses known to induce tumors in mice caused no morphologically or histopathologically observable neoplastic or nonneoplastic lesions in any of the target organs of mice. However, 7 months after cessation of exposure (recovery), urethane caused an increase in the incidence of nonneoplastic and neoplastic lesions in a number of organs. This clearly confirmed earlier findings that urethane is a potent multisite carcinogen in mice. Target organs of urethane-induced tumors included liver, lung, and Harderian gland in agreement with previous reports (NTP, 2004). In contrast, no forestomach or skin tumors were detected in mice treated with urethane in the current studies. It is likely that these differences are related to differences in the length of the dosing regimen and the route of exposure. In the current studies, urethane was administered by gavage at 1, 10, and 100 mg/kg/day for 6 weeks followed by 7 months of recovery versus 2-year drinking water exposure at 10, 30, and 90 ppm with a corresponding daily intake of 0.9, 2.7, and 8.7 mg/kg/day (NTP, 2004). In fact, lung adenomas were the only reported urethane-induced tumors in mice in earlier short-term studies (Kristiansen et al., 1990). It remains to be established if exposure to urethane for a period shorter than 6 weeks and a recovery period shorter than 7 months are sufficient to eventually lead to carcinogenesis.
Present results showed that administration of urethane for a comparatively short time (6 weeks) triggered biological changes in the target organs that were not detectable histopathologically, and that these changes continued to drive the progression of tumorogenesis during recovery, and in the absence of exposure to urethane. Earlier studies demonstrated that urethane is genotoxic (Hoffler et al., 2005; Witt et al., 2000) and this effect was dependent on CYP2E1-mediated metabolism (Hoffler et al., 2005). The potent genotoxicity of urethane in conjunction with the short onset of tumor formation suggested that it is most likely that urethane is acting as a complete carcinogen and that the initiation-promotion-progression model may explain the induction of tumors by urethane. Evidence to support the initiating potential of urethane includes the potent genotoxicity of urethane and the formation of DNA adducts (Foureman et al., 1994; Hoffler et al., 2005), which is consistent with epoxide formation. Once initiation occurs, sustained enhancement of cellular proliferation will favor clonal expansion of initiated cells and increase the probability of additional genetic damage, thereby contributing to tumor promotion and progression in the affected organs (Cohen and Ellwein, 1990; Ghanayem et al., 1994). Further, induction of sustained hyperplasia as seen in all three target organs may promote initiated cells and shorten the time available for cells to repair damaged DNA prior to replication, thereby "fixing" the initiating event (Kaufmann et al., 1987). Based on this model, urethane may be classified as a complete carcinogen.
Although there are minor variations, the pattern of urethane-induced preneoplastic and neoplastic lesions generally resembles the pattern of lesions observed in mice treated with other epoxide-forming chemical carcinogens. Chemicals such as butadiene, isoprene, chlororprene, vinyl chloride, vinyl bromide, vinyl fluoride, acrylonitrile, benzene, and styrene are metabolically activated via CYPs, primarily CYP2E1 to form epoxide intermediates (Ghanayem et al., 2000). Epoxides are three-membered cyclic ethers that are potent mutagens, reproductive toxicants, and carcinogens (Ghanayem et al., 2000). The most common targets of epoxides, such as glycidol and ethylene oxide, and epoxide-forming chemical carcinogens in mice are the liver, lung, Harderian gland, forestomach, and hemangiosarcomas of the circulatory system (Ghanayem et al., 2002; Melnick, 2002). Most of these chemicals are classified as "known human carcinogens" or "probable human carcinogens." They are strong alkylating agents that can directly react with nucleophilic sites leading to the formation of protein and DNA adducts (Melnick, 2002). Further, epoxides and epoxide-forming chemical carcinogens are known inducers of mutations in genes that regulate cell proliferation. Thus, activation of the K-ras proto-oncogene and deregulation of the p53 tumor suppressor gene are molecular changes consistent with DNA alterations caused by these chemicals (Ghanayem et al., 2000; Koskinen and Plna, 2000; Melnick, 2002). For example, point mutations in the K-ras proto-oncogene of both humans and mice (Ki-ras) at codons 12, 13, and 61 are common in many non-small cell lung cancers such as adenocarcinomas (Ichikawa et al., 1996). Indicative of urethane-induced lung tumors in mice are ATTA transversions located in the middle of the 61st codon of the Ki-ras gene. Moreover, this mutation is consistent with the DNA adduct formation by VCE, urethane's proposed ultimate carcinogenic metabolite (Ichikawa et al., 1996). Alterations in the tumor suppressor gene, p53, are the most frequently observed genetic lesions (approximately 50%) in human cancers (Miller, 1999). Studies investigating the role of p53 in rodent tumorogenesis initially suggested that rodent tumors might be similar to neoplastic lesions observed in humans with regards to altered p53 activity, especially in the liver (Yin et al., 1998). However, much debate has centered on the role of this tumor suppressor gene in the development of spontaneous and chemically induced lung tumors (Chen et al., 1993; Devereux et al., 1993; Hegi et al., 1993; Miller, 1999). Studies conducted in the lung after urethane exposure have indicated both the lack of irregular p53 activity as well as identified mutations of the p53 gene, primarily C to T and G to A transitions during the latter stages of malignant progression (Cazorla et al., 1998; Horio et al., 1996; Ohno et al., 2001).
Overall, current work has demonstrated a clear reduction of urethane-induced neoplastic lesions in the liver, lung, and Harderian gland in Cyp2e1–/– compared to Cyp2e1+/+ mice. Further, marked reduction of urethane-induced nonneoplastic lesions was observed in Cyp2e1–/– mice. Nonneoplastic lesions, such as hyperplasia in the lung, liver, and Harderian gland, and hepatic foci are markers indicative of precancer changes (Botts et al., 1999; Ghanayem et al., 1994). However, urethane exposure did result in some neoplastic and nonneoplastic lesions in Cyp2e1–/– mice. Thus, the question arises as to why inhibition of urethane-induced tumorogenesis is not complete in Cyp2e1–/– mice? Earlier reports from this laboratory demonstrated that while more than 96% of an administered dose is metabolized via CYP2E1, 
3.5% of urethane metabolism is mediated by CYPs other than CYP2E1 (Hoffler et al., 2003). Since CYPs other than CY2E1 remain fully functional in knockout mice and are capable of oxidizing urethane via epoxidation (Fig. 1), it is therefore plausible that minor metabolism is sufficient to cause the effects seen in Cyp2e1–/– mice. In agreement with this hypothesis, a slight increase in micronuclei formation was detected in Cyp2e1–/– mice treated with 100 mg urethane/kg (Hoffler et al., 2005). Further, DNA adducts indicative of urethane metabolism to VCE were detected in Cyp2e1–/– mice treated with urethane (data not shown) and support the hypothesis that inhibition of urethane tumorogenesis in Cyp2e1–/– occurs via the inhibition of its epoxidation via CYP enzymes.
The extent of inhibition in the formation of neoplastic and nonneoplastic lesions in urethane-treated Cyp2e1–/– mice varies between the liver, lung, and Harderian gland. In other words, inhibition of urethane-induced carcinogenicity in Cyp2e1–/– mice was most significant in the liver, followed by the lung and Harderian gland. Differential organ sensitivity to urethane is complex and may be dependent on many factors including the localized expression of CYPs and the subsequent in situ metabolism in that organ, the differential sensitivities between organs, the concentration of the ultimate metabolite (VCE) at the target cell, and the repair and detoxification abilities of the organ in question. While it is well known that CYPs are highly expressed in the liver and the lung, expression of these enzymes is not well investigated in the Harderian gland. Hydrolysis of epoxides via epoxide hydrolases and glutathione conjugation via glutathione S-transferases are important pathways in the detoxification of epoxides and are expected to play a role in the variation of the responses observed in various organs of mice.
In conclusion, our earlier studies using Cyp2e1–/– mice demonstrated that CYP2E1 and not esterase is the primary enzyme responsible for urethane metabolism. Subsequent studies in this laboratory demonstrated significant inhibition of urethane-induced genotoxicity and cell proliferation in Cyp2e1–/– in comparison to Cyp2e1+/+ mice. In the present studies, we have demonstrated significant reduction in urethane-induced neoplastic and nonneoplastic lesions in Cyp2e1–/– versus Cyp2e1+/+ mice. Collectively, these studies directly proved for the first time that CYPs-mediated metabolism of urethane (primarily by CYP2E1), presumably, via the formation of VCE (Fig. 1), plays a significant role in the induction of genotoxicity and carcinogenicity by this chemical.
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ACKNOWLEDGMENTS |
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I would like to thank Drs John Turnier, Ronald Herbert, and Marti Hanes for their help in the histopathological evaluation in this study. In addition, I would like to thank Dr Grace Kissling for her help with the statistical analysis of the data. I also thank Dr Frank Gonzalez for providing us with the animals to establish our breeding colony of Cyp2e1–/– and Cyp2e1+/+ mice, and Sharon Ambrose, Melissa Basabee, and others at Pathology Associates, Research Triangle Park, for their technical help. Finally, I would like to thank Drs Tom Burka, Gordon Flake, and Undi Hoffler for their constructive review of the manuscript. This research was supported by the intramural Research Program of the NIH/National Institute of Environmental Health Sciences.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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