* Nutrition and Cancer Laboratory, Pennsylvania State University, University Park, Pennsylvania 16802; Pathology Associates International, Durham, North Carolina 27709;
Laboratory of Environmental Carcinogenesis & Mutagenesis, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709;
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
Received May 17, 2004; accepted July 7, 2004
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ABSTRACT |
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Key Words: p53; Tg.AC; v-Ha-ras; antioxidant; NAC.
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INTRODUCTION |
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Two frequently used models of carcinogenesis have been the Tg.AC (v-Ha-ras) and p53 heterozygous models (French, 2004; Tennant et al., 2001
). The Tg.AC mouse possesses multiple copies of the v-Ha-ras oncogene and is considered pre-initiated, although requiring the presence of carcinogen or tumor promoter for tumor induction (Leder et al., 1990
). The p53 heterozygous mouse exhibits accelerated progression of neoplasia to malignancy by increased genomic instability (Donehower et al., 1995
). Studies have been conducted using transgenic models such as these with single genetic manipulations (Hursting et al., 2001
; Morton et al., 2002
). The inclusion of both genetic manipulations commonly noted in human cancer into a single novel bitransgenic model would be particularly useful in elaborating changes in the cancer process caused by dietary interventions from compounds such as antioxidants (Martin et al., 2002
, 2004
).
With the use of cancer-prone models, the effect of dietary components on carcinogen-induced tumor profiles can be critically investigated. For instance, antioxidants have been used widely as food additives and supplements, and are known to inhibit chemical carcinogenesis when applied before, during, and after carcinogen exposure (Kampman et al., 2003; Riboli et al., 1996
). However, emerging evidence suggests that antioxidants may exhibit paradoxical effects on tumorigenesis by adversely modifying tumor promotion and inhibition (Seifried et al., 2003
; Trewavas and Stewart, 2003
). Thus, there is a need to critically evaluate the administration of dietary constituents such as antioxidants in cancer-prone models of human cancer.
We selected N-acetyl-L-cysteine (NAC) because it is well-recognized as an antioxidant, has been used clinically for decades in treating various diseases, and is a promising chemopreventive agent (De Vries and De Flora, 1993; Flanagan and Meredith, 1991
; Pendyala and Creaven, 1995
). We used the p53 heterozygous Tg.AC (v-Ha-ras) bitransgenic mouse as a model because it possesses both the most prevalent oncogene and the most mutated tumor suppressor gene in human cancer. Our experimental design was predicated on our previously published data, as well as on reports from other investigators (Martin et al., 2004
). Briefly, we fed NAC for 28 weeks with BP exposure for the first 10 weeks to explore the tumor profile in this novel model of carcinogenesis. Our specific aims were to characterize the tumor profile of multi-organ carcinogenesis and to evaluate the effects of a well-recognized antioxidant within the context of an individual experiment.
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MATERIALS AND METHODS |
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Mice were procured at 10 weeks of age and quarantined for 2 weeks at the Association for Accreditation of Laboratory Animal Care (AAALAC)-accredited NIEHS facility prior to commencement of experiments. Mice were singly housed in temperature-controlled rooms and regulated with a 12/12 h light/dark schedule. Initially, mice (n = 30 for each of two dietary groups for each sex) were given tap water ad libitum and placed on either a basal or a semipurified diet containing 20% soy protein or basal diet plus 3% NAC (Research Diets, New Brunswick, NJ) for 2 weeks prior to dosing with benzo(a)pyrene (BP) (CAS 50328; Aldrich, Milwaukee, WI). Each sex was further subdivided to produce four groups per sex (2 x 2 design), which received corn oil vehicle alone or BP (20 mg/kg body weight) in corn oil for 10 weeks, given twice per week. The BP solutions were prepared fresh each week, and the dosage was calculated from the individual animal body weight from that week. After 10 weeks, dosing was discontinued and mice remained on the respective diets for subsequent observation for the succeeding 18 weeks.
Mice were weighed weekly and food intake was determined to monitor the onset of morbidity and toxicity. Mice were also evaluated daily for visible or palpable lesions. The criteria for unscheduled sacrifice of animals prior to 28 weeks were the presence of gross masses, morbidity, or body weight loss 20% of mouse maximal weight. Tissues from some mice (9 males, 4 females) that died prior to 28 weeks were not collected because of marked autolysis. All procedures and studies involving animals were reviewed and approved by the NIEHS Animal Care and Use Committee.
Histology. Selected tissues were excised, trimmed, and immediately placed in 10% neutral buffered formalin (pH 7.0) for 24 h, followed by transfer to 70% ethanol for 24 h. The fixed tissues were embedded in paraffin, sectioned (5 µm), and stained with hematoxylin and eosin for histopathological analysis. Collected tissues included the thymus, heart, lungs, stomach, mesenteric lymph node, spleen, kidney, testis/ovary, and urinary bladder. Gross lesions from nonselected sites were collected if present in the skin, mammary and salivary glands, or thoracoabdominal cavities. Gross lesions in other tissue sites were not collected.
Statistics. Differences in regression were assessed by Fisher's exact test. Differences in the incidences of tumors between the four dietary groups were tested by chi-square analysis. Survival differences were determined by life-table analysis. Differences in maximum tumor yields between groups were analyzed by analysis of variance (ANOVA).
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RESULTS |
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In examined tissues of females, increased incidences of thymic lymphoid hyperplasia (8/22) were observed in the two groups exposed to BP compared to controls fed the basal diet or the NAC-treated diet, but without BP exposure (2/21) (Table 1). The BP+NAC group (5/11) appeared to have a greater incidence of thymic lymphoid hyperplasia than the BP group (3/11) when compared to controls (1/11). Males in all groups exhibited a 15% (6/40) incidence of thymic lymphoid hyperplasia, similar to the incidence in untreated female controls (Table 2). Thymic lymphoid atrophy and the incidence in thymic cysts appeared in both sexes but slightly more often in females than males. Interestingly, in males, neither lesion appeared in the BP-exposed groups.
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Because antioxidants can reduce the incidence of gastric lesions when given with carcinogens, both the glandular stomach and the forestomach were examined for pathologic abnormities (Table 3). In males, BP-induced forestomach papillomas occurred in 63% (5/8) of mice on diets without NAC and were reduced to 14% (1/7) with NAC in the diet. There was no difference between the vehicle-treated basal and NAC-fed groups. In contrast, females fed NAC, but not dosed with BP, displayed a 62% (8/13) incidence of forestomach papillomas similar to incidences in BP-dosed mice fed either diet. Females displayed approximately twice the incidence of cumulative forestomach papillomas as male mice (28/53, 53% versus 8/26, 31%), respectively.
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DISCUSSION |
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Unexpectedly, NAC reduced mean body weight by 15% in males but did not impair food intake or increase mortality. Although not significant, this effect could be biologically relevant. Other investigators using standard carcinogenesis protocols have noted that modest changes in body weight can significantly affect survival and tumor incidence in mice. In fact, over 50% of the variation in liver tumor incidence in mice from numerous studies could be ascribed to increases in body weight (Turturro et al., 1996). In a report using Tg.AC mice, a nontoxic, water-soluble antioxidant from spinach, delivered either topically or orally, reduced final body weights of mice by
16% compared to control mice (Nyska et al., 2001
).
N-acetyl-L-cysteine and BP appeared to exhibit different but discernible effects on survival in this model. While all groups of female mice exhibited a linear decline in survival to 30%, male mice exhibited different kinetics of survival. During BP exposure, survival in male mice declined, but upon cessation of carcinogen exposure it remained constant from week 12 to week 26. In males, BP-treatment reduced survival further compared to controls, suggesting BP-induced toxicity. The mean spontaneous survival times in p53 heterozygous Tg.AC bitransgenic mice have been reported to be greater for males than for females, with 50% mortality occurring at 37 and 30 weeks, respectively (Moser et al., 2000
). We have previously reported that dietary NAC significantly increases survival of male bitransgenic mice, but notably decreases survival in female mice when BP is administered according to a skin paint protocol (Martin et al., 2001
, 2002
).
Benzo(a)pyrene exposure induced many carcinogen-specific effects. For instance, the stomach is a key target for BP delivered by gavage (Hakura et al., 1998), and it is also a frequent target in Tg.AC mice where spontaneous background lesion formation occurs at a rate of
9% (Mahler et al., 1996
). It is noteworthy that in the bitransgenic model described here, we observed an overall 29% incidence of gastric papilloma formation in the control group of female mice given corn oil alone that was threefold the spontaneous background incidence reported for Tg.AC mice. For females, the background incidence in the control group was half that of the other three groups receiving NAC and/or BP, suggesting a chemical-dependent effect. Nonspecific irritation of the stomach in ras expressing mice can increase lesion formation (Hansen et al., 1996
; Mahler et al., 1998
). At necropsy we observed compaction and pelleting of diet in the stomachs of mice. In male mice, BP increased (42%, 5/8) papilloma formation more than twofold above background, which was attenuated to 19% (1/7) with NAC addition. This observation is consistent with the results of other investigators who have shown antioxidant-dependent decreases in carcinogen-induced gastric papillomas (Badary et al., 1999
; Deshpande et al., 1997
). The lack of a BP-induced effect or protection by NAC in female mice is likely due to the high background of tumor incidence.
An unusual but naturally occurring neoplasm in Tg.AC mice is erythroleukemia, which does not occur in the FVB/N parental strain (Mahler et al., 1996). However, the occurrence of erythroleukemia, as well as other hematoproliferative disorders, is associated with mutant ras transgene expression. The spontaneous background incidence of erythroleukemia is 14% in male and female mice with either p53 heterozygosity or the Tg.AC genotype (Mahler et al., 1998
). Although we did not observe lesions in three of the male groups (n = 38), erythroleukemia did occur in 25% (3/12) of the mice in the BP + NAC group. Although the sample size is low, the lack of lesions in the other three groups suggests an interaction of diet and sex.
We noted two other potentially detrimental effects in this mouse model. Thymic lymphoid hyperplasia occurred in 10% (1/11) of female control mice and was increased to 27% (3/11) by BP exposure. This further increased to 45% (5/11) in mice treated with BP and fed NAC, suggesting an exacerbating effect between the two. A clear limitation in this study is the small number of samples (11 lesions from 15 mice) available for analysis. We also noted in female control mice a 29% (4/14) incidence of forestomach papilloma, which was increased to 62% (8/13) in mice fed NAC but not treated with BP. Treatment with BP either alone or in combination with NAC did not further increase this incidence. The high background level of hyperplasia in female mice precluded any conclusions regarding a protective effect.
The combination of BP and NAC exposure in a cancer-prone model establishes an environment conducive to pathology. Benzo(a)pyrene is metabolized actively to reactive metabolites in the liver, which undergo enterohepatic recirculation, thus increasing the duration of exposure and potential for DNA damage (Viau et al., 2000). Studies in mice demonstrate that oral dosing with BP increased the mutational frequency in proliferative tissues in the order of forestomach, spleen, liver, and kidney (Viau et al., 2000
). Hepatic midzonal hypertrophy was observed in nearly 100% of all mice. In addition to the effects of BP, NAC can accumulate in the liver and adversely affect BP bioactivation in mice and alter glutathione levels (De Flora et al., 1985
; McLellan et al., 1995
). Both male and female mice were susceptible to hepatic midzonal hypertrophy and hepatic cytoplasmic vacuolization (54% and 92%, respectively). Interestingly, however, the females were more susceptible to increased hematopoietic cell proliferation and mixed cell infiltrates, which could have contributed singly or in combination to decreased survival. Necrosis also appeared in the livers of female mice after BP exposure, but not in untreated controls, supporting BP-induced cytotoxicity.
Splenic hepatopoietic cell proliferation was higher in females (60%, 32/53) compared to males (14%, 7/50) supporting sex-specific sensitivity. Although HCP occurred with similar frequencies in male groups, erythroleukemia was localized to the BP + NAC group. In males, BP also increased the number of malignant lymphomas (33%, 4/12), as expected, and the incidence was reduced with NAC inclusion (17%, 2/12). Malignant lymphoma occurred with incidences of 731% in female mice fed NAC and/or exposed to BP, but no lymphomas occurred in the control group. Our prior studies in this model had suggested a NAC-dependent appearance of lesions consistent with lymphoma (Martin et al., 2001). Thus, the list of potential target organs was specifically selected to monitor a wide range of potential systemic effects of BP exposure and to allow comparison with results obtained in independent studies of p53 and Tg.AC.
The reason for the observed differences between male and female mice is unclear. Glutathione levels and glutathione transferase activities, as well as enzymes involved in carcinogen metabolism (i.e., cytochrome P450s), and detoxification are reportedly higher in male mice, rendering them presumably more efficient then females at metabolizing and inactivating BP and/or its metabolites (Sharma et al., 1997; Singh et al., 1998
). Substantial sex-dependent differences also exist in gene expression involved in signaling, growth control, transcription, and other pathways, as well as upregulation of both G proteincoupled and nuclear receptors, upon acute and chronic carcinogen exposure (Kovalchuk et al., 2004
). Moreover, the effects of androgen and estrogen, and their metabolites, are also a consideration in neoplasia. Thus, the mechanism(s) for sex-specific observations is unclear and likely complex and multifactorial.
Accumulating evidence supports the potential for antioxidants to exhibit paradoxical actions in tumorigenesis (Trewavas and Stewart, 2003). Although the Tg.AC and p53 heterozygous mouse models have been characterized independently, validation of this bitransgenic model similar to other bitransgenic models that have emerged for cancer studies is lacking (Hursting et al., 1999
; MacLeod and Jacks, 1999
). For example, the MMTV-Wnt1/MMTV-Neu and MMTV-Wnt1/p53-/-models were designed to study activating mutations of ras in association with other gene mutations (Podsypanina et al., 2004
). Other investigators have combined the Tg.AC genotype with the Werner syndrome gene (Wrn) to explore the interaction and effect of mutant ras expression with Wrn on genomic stability (Leder et al., 2002
). Although a clear limitation of the data presented here is the lack of any statistically significant effects because of the relatively small sample size, the data do further characterize this novel model and suggest that additional, larger studies should be conducted to validate its usefulness in short-term dietary and carcinogenicity studies.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Nutrition & Cancer Laboratory, Pennsylvania State University, 126 Henderson Building South, University Park, PA 16802. Fax: (814) 863-6103. E-mail: krm12{at}psu.edu.
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