Mutagenesis, Vol. 18, No. 1, 7-12,
January 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
Mutagenesis and DNA adduct formation in the mouse mammary gland exposed to 2-hydroxyamino-1-methyl-6-phenylimidazo-[4,5-b]pyridine in whole organ culture
Chemical Carcinogenesis Section, Laboratory of Experimental Carcinogenesis, National Cancer Institute, Center for Cancer Research, Bethesda, MD 20892, USA and 1 Department of Pathology, Medical College of Ohio, Toledo, OH 43614, USA
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Abstract |
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2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a mutagen and rodent mammary gland carcinogen found in the human diet. 2-Hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-hydroxy-PhIP) is the proximate reactive metabolite of PhIP associated with PhIP–DNA adduct formation and mutagenesis. In the current study, whole mammary glands obtained from transgenic C57Bl/6 mice carrying the plasmid–lacZ mutational reporter gene were cultured in defined medium and exposed to various concentrations of N-hydroxy-PhIP for 24 h. At various times after N-hydroxy-PhIP exposure, PhIP–DNA adduct levels were determined by the 32P-post-labeling assay and the lacZ- mutant frequency determined by the positive selection system. Glands were cultured in either medium containing insulin (I medium), necessary for maintenance of the gland, or I medium containing prolactin, aldosterone and hydrocortisone (IPAH medium) to induce lobuloalveolar development. At 3 and 7 days after exposure to 10 µM N-hydroxy-PhIP, mutant frequency was upwards of 9-fold higher in glands incubated in IPAH medium than in I medium (15.2 ± 1.9 and 1.6 ± 0.7x10-3, respectively, 3 day time point). PhIP–DNA adduct levels were 1.7-fold higher in glands cultivated in IPAH medium than in I medium immediately after exposure to 10 µM N-hydroxy-PhIP. A statistically significant reduction in PhIP–DNA adduct levels occurred with time in glands cultivated in IPAH medium but not I medium (one-way analysis of variance, P < 0.05). By 7 days after exposure, PhIP–DNA adduct levels were similar in glands cultured in I and IPAH medium (3.2 ± 0.2 and 2.8 ± 0.29 adducts/107 nucleotides, respectively). DNA synthesis as measured by [3H]thymidine labeling was
2-fold higher in glands cultured in IPAH medium than in I medium. The higher mutant frequency in glands cultivated in IPAH medium versus I medium appeared to be due to a combination of higher initial PhIP–DNA adduct levels and a greater fixation of mutations that occurred at higher proliferation rates. The findings indicate that mammotrophic hormones influence the mutagenicity of PhIP in the mammary gland in vitro and emphasize the importance of hormonal milieu on carcinogen–DNA adduct-induced mutations in this organ. |
Introduction |
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The food-derived heterocyclic amines (HCAs) comprise a group of mutagens/carcinogens produced during the cooking of meats, including beef, chicken and fish (Sugimura, 1997
). One HCA, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is currently recognized as the principal carcinogenic HCA in the human diet (Layton et al., 1995; Nagao, 1999; Sinha et al., 2000). PhIP has been shown to be a mammary gland carcinogen in rats and mice (Ito et al., 1991; Snyderwine et al., 1998; Nagao et al., 2002). Population studies have further indicated that well-done cooked meat consumption and PhIP exposure may increase the risk of human breast cancer (Zheng et al., 1998; Sinha et al., 2000).
PhIP is a procarcinogen that requires metabolic activation for mutagenicity and carcinogenicity (Schut and Snyderwine, 1999). The major route of metabolic activation of PhIP involves cytochrome P450-mediated N-hydroxylation followed by phase II esterification by enzymes such as N-acetyltransferase. Reactive esters form guanine adducts, the principle adduct being N-(deoxyguanosin-8-yl)-PhIP. PhIP–DNA adducts have been shown to induce mutations in several in vitro assays (Carothers et al., 1994; Endo et al., 1994; Shibutani et al., 1999). In addition, recent studies using Big Blue rats, transgenic animals carrying the lacI mutational reporter gene, have shown that PhIP induces mutations in the mammary gland (Okochi et al., 1999; Yu et al., 2002), however, detailed studies on the role of PhIP–DNA adducts and cell proliferation on mutation induction in the mammary gland have not yet been carried out.
The mouse mammary gland whole organ culture assay is a well-established culture system used for studies on hormonal regulation of mammary gland growth and differentiation and for studies of epithelial–stromal interactions (Ichinose and Nandi, 1964, 1966; Lin et al., 1976; Ginsburg and Vonderhaar, 2000; Mehta, 2000). Ichinose and Nandi (1964, 1966) first showed that whole mammary glands from hormone-pretreated pubescent mice can undergo normal lobuloalveolar development in vitro in the presence of specific lactogenic hormones. The survival of the glands in serum-free defined medium depended on insulin and lobuloalveolar development and differentiation was achieved in the presence of insulin, prolactin, hydrocortisone and aldosterone. Mouse mammary gland whole organ cultures have been used to examine chemical carcinogenesis and the processes involved in neoplastic transformation in the mammary gland (Banerjee et al., 1974; Lin et al., 1976; Tonelli et al., 1979; Chatterjee and Banerjee, 1982; Mehta and Moon, 1986). In whole organ culture known mammary carcinogens such as 7,12-dimethylbenz[a]anthracene (DMBA) and N-nitrosomethylurea have been shown to cause neoplastic transformation of mammary gland epithelial cells (Telang et al., 1979; Iyer and Banerjee, 1981; Delp et al., 1990).
The whole organ culture assay provides an ideal system to examine the influence of carcinogen–DNA adducts and hormonal milieu on mutagenesis in the mammary gland. No previous study has used this model to study the relationship between carcinogen–DNA adduct levels and the induction of mutations. Inducing a range of carcinogen–DNA adduct levels in organ culture is relatively facile. The organ culture assay bypasses the variables that can affect adduct formation in the mammary gland in vivo, such as carcinogen absorption, distribution, metabolic activation and detoxification. In addition, in whole organ culture development of the gland can be experimentally controlled by the hormone composition of the medium. Therefore, mutagenesis can be studied in whole organ culture in the context of normal mammary gland physiology and under defined conditions for growth and differentiation.
The current study describes the use of the mouse mammary gland whole organ culture assay for studies on the mutagenicity of PhIP. Glands were obtain from transgenic mice carrying the plasmid–lacZ mutational reporter gene (Boerringter et al., 1995) and mutagenesis examined under different hormonal conditions. These studies were undertaken to provide insight into the factors impacting on the mutagenicity of PhIP in the mammary gland.
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Materials and methods |
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Chemicals and medium
Bovine insulin and Waymouth's MB 752/1 medium were purchased from Gibco (Invitrogen, Grand Island, NY). Prolactin, aldosterone and hydrocortisone were obtained from Sigma Chemical Co. (St Louis, MO). Avertin (2,2,2-tribromoethanol) was purchased from Aldrich Chemical Co. (Milwaukee, WI). 2-Hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-hydroxy-PhIP) (purity 95.9%) was prepared by SRI International (Menlo Park, CA). [Methyl-3H]thymidine (specific activity 50 Ci/mmol) was purchased from New England Nuclear (Boston, MA).
Animals
C57Bl/6-TgN(LacZpl)60Vij mice were purchased from Jackson Laboratory (Bar Harbor, ME) and bred and housed in an AAALAC accredited facility at the National Cancer Institute (NCI). Animal housing, care and treatment were in accordance with NIH guidelines with protocols reviewed and approved by the NCI Animal Care and Use Committee. Mice were provided laboratory chow and water ad libitum and maintained on a 12 h light/12 h dark cycle throughout the study.
Organ culture
Organ culture was carried out essentially as described by Ginsburg and Vonderhaar (2000). Briefly, 22–24 day old female mice were anesthetized with Avertin and one 21 day release hormone pellet containing 17ß-estradiol (0.01 mg) and progesterone (10 mg) (Innovative Research of America, Sarasota, FL) was implanted in the intrascapular area. After 14 days, the fourth mammary gland was removed from the mice, supported on siliconized lens paper and placed in culture. Culturing was carried out with Waymouth's MB 752/1 medium supplemented with penicillin/streptomycin and 5 µg/ml bovine insulin (I medium) or in medium also containing 5 µg/ml prolactin, 1 µg/ml aldosterone and 1 µg/ml hydrocortisone (IPAH medium). Glands were incubated at 50% oxygen, 5% CO2 in humidified air at 37°C (Heraeus Instruments, Newtown, CT). N-hydroxy-PhIP was dissolved in dimethylsulfoxide (DMSO) and added to the medium. The final concentration of DMSO in the incubation medium was 0.022%. Glands were cultivated in IPAH medium or I medium for 72 h and then treated with N-hydroxy-PhIP for 24 h. The medium was then removed and the glands washed three times with phosphate-buffered saline, pH 7.4 (PBS). Glands were either collected after N-hydroxy-PhIP exposure or further cultivated in medium for up to 7 days prior to DNA isolation.
Cell proliferation assay
Cell proliferation was assayed using the method described previously (Lin et al., 1976) with minor modifications. Mammary glands in organ culture were incubated with 5 µCi/ml [methyl-3H]thymidine for 4 h before collecting tissue. At the end of the incubation, glands were washed with PBS three times and stored at –80°C until use. The glands were homogenized in 2 ml of cold 5% trichloroacetic acid and the homogenates centrifuged at 12 000 g for 20 min. The supernatant was discarded and 1 ml of 10% trichloroacetic acid was added to the resulting pellet. The sample lysate was vortexed vigorously and incubated at 70°C for 30 min. The radioactivity in a 200 µl aliquot of the lysate was measured by scintillation spectrometry and then normalized to the protein concentration, which was determined by the Bradford method (Bradford, 1976).
Whole mount fixation
Whole mount fixation was carried out as described previously (Delp et al., 1990). Briefly, glands were rinsed with PBS, lightly blotted and placed in tissue fixative containing glacial acetic acid and ethanol. Glands were subsequently stained with carmine alum and, following washing in ethanol and toluene, mounted on glass slides. Glands were visualized under a stereomicroscope.
DNA extraction
Organ cultured mammary glands were rinsed in PBS and high molecular weight genomic DNA was isolated by phenol/chloroform extraction as described previously (Vijg and Douglas, 1996). The DNA concentration and purity were assessed spectrophotometrically. Two mammary glands were routinely pooled for each DNA sample and DNA was used for the mutation assay and the 32P-post-labeling assay.
lacZ mutant assay
The lacZ mutant assay is based on that described previously (Boerringter et al., 1995; Dolle et al., 1996; Vijg and Douglas, 1996) and was carried out following the methodology and using the reagents provided by Leven Inc. (Bogart, GA). This procedure involved rescue of the pUR288 plasmid from the purified genomic DNA and determination of lacZ- mutant frequencies using a phenyl-ß-D-galactoside (P-gal) positive selection system. Briefly, genomic DNA was digested with HindIII and plasmid pUR288 harboring the lacZ gene recovered by immunomagnetic plasmid separation. Plasmids were recircularized, precipitated and electroporated into Escherichia coli C (lacZ-/galE-) indicator cells. After recovery of the E.coli, a 1/1000 part dilution was plated on LB medium containing 5-bromo-4-chloro-3-indolyl-ß-D-galactoside (X-gal) to determine rescue efficiency. The remainder was plated on medium containing P-gal to select for mutant clones. The mutant frequency was determined as (number of colonies on the P-gal plate)/(number of colonies on the X-gal platex1000).
32P-post-labeling assay
PhIP–DNA adducts were assayed by the post-labeling method, which resolves [32P]ATP-labeled bisphosphonucleotide adducts as spots on autoradiograms following chromatography on polyethyleneimine cellulose sheets. The assay was carried out under intensification conditions as previously described (Schut and Herzog, 1992). PhIP–DNA adduct levels were quantified through analysis of an authentic adduct standard generating a standard curve relating relative adduct labeling values to adduct levels per 107 nucleotides (Yu et al., 2002).
Statistical analysis
Statistical analysis was carried out using SigmaStat statistical software v.2.0 (Jandel Scientific Software, San Rafael, CA).
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Results |
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Experiments were first carried out to examine the concentrations of N-hydroxy-PhIP required for DNA adduct formation and induction of mutations in whole organ culture. Mammary glands were incubated with various concentrations of N-hydroxy-PhIP in IPAH medium for 24 h prior to DNA adduct analysis. PhIP–DNA adducts were detected in cultured mammary glands incubated with 5, 10 and 20 µM N-hydroxy-PhIP, but not with 1 µM N-hydroxy-PhIP (Figure 1
). The profile of PhIP–DNA adducts in the mouse mammary gland in organ culture was the same as that found in the mammary gland of rats that succumb to the carcinogenic effects of PhIP (Ghoshal et al., 1995). Adduct levels increased approximately linearly with concentration and reached 17.8 ± 3.9 adducts/107 nucleotides after exposure to 20 µM N-hydroxy-PhIP (mean ± standard error, n = 4). Mutant frequency was examined after an additional 7 days in culture (Figure 2). The 7 day time point provided an optimum manifestation time (Heddle, 1999) for mutagenesis. Preliminary studies revealed that mutant frequency was low just after N-hydroxy-PhIP exposure and increased with continued culturing of the gland after N-hydroxy-PhIP removal for up to 1 week. In accordance with PhIP–DNA adduct levels being non-detectable at 1 µM N-hydroxy-PhIP by 32P-post-labeling analysis (Figure 1), mutant frequency at this concentration was not significantly different from the mutant frequency found in control cultures not exposed to carcinogen (control values ranged between 2 and 8x10-4). Mutant frequency increased as the concentration of N-hydroxy-PhIP increased to 5 and 10 µM (Figure 2). Although PhIP–DNA adduct levels were 2.3-fold higher at 20 than 10 µM N-hydroxy-PhIP, mutant frequency was similar at these two concentrations. Whole mount analysis of the mammary glands 7 days after exposure to 20 µM N-hydroxy-PhIP revealed relatively poor lobuloalveolar development in comparison to glands exposed to lower concentrations of N-hydroxy-PhIP. The 10 µM concentration of N-hydroxy-PhIP, which maximized mutant frequency, was used in further studies comparing the effects of I medium and IPAH medium on PhIP–DNA adduct levels and mutant frequency.
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Consistent with prior reports (Ichinose and Nandi, 1966
; Lin et al., 1976; Ginsburg and Vonderhaar, 2000), whole mount images showed lobuloalveolar development in the mammary glands cultivated in IPAH medium whereas development was sparse in glands in I medium (Figure 3). Mutant frequency in the mammary glands cultivated in I and IPAH medium was examined at 3 and 7 days after treatment with 10 µM N-hydroxy-PhIP (Figure 4). At both time points, mutant frequency was higher in glands incubated in IPAH medium than in I medium. At the 3 and 7 day time points, mutant frequency was respectively 9- and 5-fold higher in glands incubated in IPAH medium than I medium. In either I or IPAH medium the mutant frequency was found to increase with time in culture from 3 to 7 days, a finding that illustrates the impact of manifestation time on mutant frequency.
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To investigate the possible mechanisms for the higher mutant frequency in mammary glands cultivated in IPAH medium, PhIP–DNA adduct formation and removal was examined in glands cultivated in IPAH and I medium (Figure 5
). Immediately after a 24 h exposure to 10 µM N-hydroxy-PhIP, PhIP–DNA adduct levels were 1.7-fold higher in glands cultivated in IPAH medium than in I medium. However, 1 day after exposure adduct levels were significantly lower in glands in IPAH medium than in glands in I medium (Student's t-test, P < 0.05). After 7 days, similar adduct levels were found in organs cultivated in either medium. Interestingly, there was a statistically significant reduction in PhIP–DNA adduct levels over time in glands cultivated in IPAH medium (one-way analysis of variance, P < 0.05), however, no statistically significant change in adduct levels occurred with time when glands were cultivated in I medium.
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Proliferation was also examined in glands in I and IPAH medium and exposed to N-hydroxy-PhIP (Figure 6
). DNA synthesis measured by [3H]thymidine labeling was 2-fold higher in glands cultured in IPAH medium than in I medium. N-hydroxy-PhIP at the 10 µM concentration did not significantly alter proliferation in glands cultured in either medium, supporting the proposal that this concentration was neither toxic nor growth promoting to glands in culture.
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Discussion |
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In the current study the mouse mammary gland organ culture assay was used to study PhIP–DNA adduct formation and mutagenesis in the mammary gland. N-hydroxy-PhIP was found to form PhIP–DNA adducts in a concentration-dependent manner in the mammary gland in organ culture. At concentrations of 1, 5 and 10 µM N-hydroxy-PhIP, the mutant frequency was observed to increase concomitant with PhIP–DNA adduct levels. These data support a direct relationship between adducts and mutation induction in this system. It is notable, however, that as the concentration of N-hydroxy-PhIP increased from 10 to 20 µM the doubling in PhIP–DNA adduct levels was not associated with a significant increase in mutant frequency. Since 20 µM N-hydroxy-PhIP was observed to retard lobuloalveolar development, it appeared that N-hydroxy-PhIP was toxic to the gland at this concentration. This toxicity may have accounted for the relatively low mutant frequency despite high PhIP–DNA adduct levels at 20 µM N-hydroxy-PhIP.
It is well recognized that hormones influence mammary gland carcinogenesis (Russo,I.H. and Russo,J., 1998). Whole organ culture assay using glands from plasmid–lacZ mice provides a means to study the mechanism of action of specific mammotrophic hormones on mammary gland mutagenesis. The current study examined the differences in mutant frequency in medium containing only insulin (I medium) and medium that contained additional hormones required for mammary gland growth and development. Insulin is required for survival of the mammary gland in organ culture and is a permissive factor in the response to mammotrophic hormones, but in I medium development and differentiation of the gland is minimal (Ichinose and Nandi, 1964, 1966; Mukherjee et al., 1973). With the further addition of prolactin, aldosterone and hydrocortisone, lobuloalveolar development was clearly observed in our cultures (Figure 3
). Mutant frequency was significantly higher in IPAH medium than in I medium, indicating that hormonal factors that influence development were also associated with higher mutagenesis. The higher mutant frequency in the glands cultured in IPAH appeared to be partly associated with higher initial PhIP–DNA adduct levels. Following exposure to N-hydroxy-PhIP, adduct levels in glands cultured in IPAH medium were 1.7-fold higher than in glands cultured in I medium. It is possible that the higher adduct levels in glands in IPAH medium are a consequence of a greater capacity for phase II metabolic activation in glands cultured in IPAH medium in comparison to I medium. Our previous studies with the mammary epithelial cell line MCF10A have also suggested that serum and growth factors potentially influence phase II metabolic activation (Venugopal et al., 1999). PhIP—DNA adduct levels, however, appear to only partially explain the differences in mutant frequency between I and IPAH medium, since initial adduct levels differed by <2-fold while mutant frequency varied by >9-fold. In addition, whereas there was a significant reduction in adduct levels over time in glands cultured in IPAH medium, little to no removal of adducts was seen in glands cultured in I medium over the 1 week period after N-hydroxy-PhIP exposure. After 1 week in culture adduct levels were similar in glands cultured in IPAH and I medium while mutant frequency still differed by 5-fold. Despite the potential repair of PhIP–DNA adducts in IPAH medium and apparent limited repair in I medium, mutant frequency was considerably higher in glands cultured in IPAH medium.
Another factor potentially influencing mutant frequency in specific tissues is the rate of cell proliferation (Heddle, 1999). Proliferation has been shown to be required for both mutation and DNA repair (Bielas and Heddle, 2000), and the findings from the current study are consistent with this concept. In the mammary gland in organ culture proliferation was 2-fold higher in IPAH medium than in I medium. A higher rate of proliferation in glands cultured in IPAH medium is likely to increase the fixation of PhIP–DNA adducts as mutations. Therefore, it appears likely that the higher mutant frequency in IPAH medium is due to a combination of higher initial PhIP–DNA adduct levels and greater fixation of mutations. Whether the mammotrophic hormones affect other processes impacting on carcinogen–DNA adduct-induced mutagenesis in the mammary gland requires further study. The observation that hormones influence the mutagenicity of PhIP in the mammary gland in vitro emphasizes the importance of hormonal milieu in carcinogen–DNA adduct-induced mutations in this organ.
Hormonal factors that influence mammary gland growth and differentiation modify susceptibility to chemical carcinogenesis (Sinha and Pazik, 1981; Russo,J. and Russo,I.H., 1987; Russo,I.H. and Russo,J., 1998). Previous studies suggest that specific hormones affect neoplastic transformation in the mammary gland in whole organ culture and the formation of nodular preneoplastic lesions detected in organ culture (Lin et al., 1976). The frequency of nodular preneoplastic lesions induced by DMBA in organ culture has been shown to be highest in IPAH medium and to be associated with a high rate of cell proliferation (Lin et al., 1976). The findings from the current study are consistent with the notion that IPAH medium may in part facilitate development of preneoplastic lesions via an increase in carcinogen-induced mutagenesis.
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Acknowledgments |
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The authors thank Dr Barbara Vonderhaar and Mrs Erika Ginsburg (National Cancer Institute) for instruction on the mouse mammary gland whole organ culture assay and Dr Klaus Felix (National Cancer Institute) for helpful suggestions with the plasmid–lacZ mutation assay.
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Notes |
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2 To whom correspondence should be addressed at: Chemical Carcinogenesis Section, Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, Building 37, Room 3C28, 37 Convent Drive, MSC 4258, Bethesda, MD 20892-4258, USA. Tel: +1 301 496 5688; Fax: +1 301 496 0734; Email: elizabeth_snyderwine{at}nih.gov
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Received on May 17, 2002; revised on June 26, 2002; accepted on June 26, 2002.
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