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Carcinogenesis Advance Access originally published online on February 14, 2008
Carcinogenesis 2008 29(4):866-874; doi:10.1093/carcin/bgn030
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Mgmt deficiency alters the in vivo mutational spectrum of tissues exposed to the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

Linda E. Sandercock, Jennifer N. Hahn, Li Li1, H.Artee Luchman, Jennette L. Giesbrecht, Lisa A. Peterson1 and Frank R. Jirik*

Department of Biochemistry and Molecular Biology, The McCaig Institute for Bone and Joint Health, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta, Canada T2N 4N1
1 Division of Environmental Health Sciences and the Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA

* To whom correspondence should be addressed. Tel: +403 220 8666; Fax: +403 210 8100;Email: jirik{at}ucalgary.ca


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
It has been proposed that O6-methylguanine DNA methyltransferase (MGMT) gene silencing in premalignant lesions and cancers of the lung might result in the acquisition of a ‘mutator’ phenotype. Previously, however, we found that Mgmt–/– mouse DNA failed to show an increase in spontaneous mutations. We thus hypothesized that only during exposure to specific environmental carcinogens would the consequences of MGMT deficiency become evident. Metabolism of the tobacco-derived nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) generates alkylating species that can react with the O6 position of deoxyguanine, thereby yielding substrates for MGMT-mediated repair. To investigate how MGMT might regulate the mutational effects of NNK, Mgmt–/– mice were crossed with a lacI-based transgenic reporter line (Big BlueTM) thus enabling an assessment of the in vivo mutagenic effects of this agent. We observed the induction of a complex spectrum of NNK-dependent lacI mutations in both control and Mgmt–/– tissues, but only a trend in the mutant frequency increases that could be attributed to MGMT deficiency. The mutational spectra of NNK-treated Mgmt–/– lungs revealed an increase in the absolute number of G:C to A:T changes accompanied by a shift in these from CpG to GpG sites, consistent with an SN1 alkylation mechanism. In keeping with the high levels of MGMT expressed in the liver, more pronounced mutagenic effects and greater differences in O6 position of deoxyguanosine adduct levels following NNK were observed in Mgmt–/– versus wild-type mice. Extrapolating to humans, MGMT-deficient cells would likely exhibit an increased mutational burden, but only following exposures to specific environmental mutagens such as NNK.

Abbreviations: dG, deoxyguanosine; dT, deoxythymidine; MCA, Monte Carlo estimation of the P-value of the hypergeometric test; Mf, mutation frequency; MGMT, O6-methylguanine DNA methyltransferase; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; O6-mG, O6-methylguanine; O6-pobG, O6-[4-oxo-4-(3-pyridyl)butyl]guanine; WT, wild-type


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Among the numerous mutagens present in tobacco smoke, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), formed during the tobacco curing process, is viewed as one of the most potent lung carcinogens (1). In rodents, NNK can induce lung tumors irrespective of the route of administration, due to the metabolic activation of NNK by specific cytochrome P450 oxidoreductase isoforms expressed in the lung (2), as well as to genetic predispositions in mice that lead to lung neoplasms even after a single dose of NNK. Thus, differences in susceptibility to NNK-induced lung adenomas exist among inbred mouse strains, for example, with A/J being much more susceptible than C57BL/6 (3–5). Not only is NNK a potent lung carcinogen in rodent models (4), but almost certainly in humans as well, according to an International Agency for Research on Cancer working group (6). This agent is metabolized in several ways, generating alkylating species that react with sites on various DNA bases, including the O6 position of deoxyguanosine (O6-dG) (7). O6-methylguanine (O6-mG) levels, in turn, play a part in determining the extent of NNK-induced rodent lung tumorigenesis (4,8). In addition to generating O6-dG alkylations, other NNK metabolites pyridyloxobutylate DNA bases, generating lesions that can lead to both G:C to A:T and G:C to T:A mutations (3,9,10). DNA single-strand breaks and increased levels of 8-oxo dG, as a result of NNK-induced oxidative stress, have also been reported (11).

O6-mG and the pyridyloxobutyl adduct, O6-[4-oxo-4-(3-pyridyl)butyl]guanine (O6-pobG), are substrates for the DNA repair protein O6-methylguanine DNA methyltransferase (MGMT) (12–14). MGMT (also referred to as O6-alkylguanine DNA alkyltransferase) is unique among DNA repair molecules, in that it directly removes base adducts with 1:1 stoichiometry (12). MGMT specifically removes alkyl adducts from the O6 position of dG, although O4-methyl-deoxythymidine (dT) is also a weak substrate. Left unrepaired, O6-mG and O6-pobG, O4-methyl-dT and O6-chloro-ethylguanine can lead to G:C to A:T transitions, A:T to G:C transitions and inter-strand cross-links, respectively. The latter lesion may also lead to G:C to T:A transversions upon resolution through alternate repair pathways (15). Although the physiological role of MGMT remains enigmatic, its evolutionary conservation (e.g. from archaebacteria to mammals) implies an important protective function against environmental and/or endogenously generated alkylating species (16). MGMT has also become important in oncology, owing to its significance in cancer prevention and to therapeutic regimens involving alkylating agents (17). MGMT deficiency renders animals more susceptible to alkylating agent-induced damage (17,18), whereas MGMT over-expression not only prevents alkylating agent-induced cancers in animals but also protects non-malignant tissues against alkylating agent-induced toxicities, such as bone marrow suppression (17).

In lung cancer, silencing of the MGMT gene by promoter methylation is seen in 29% of individuals with non-small cell lung cancer (19). Furthermore, MGMT promoter methylation also occurs in pre-neoplastic disease, specifically, in the DNA of shed epithelial cells present in the sputum of heavy smokers and former smokers (20–22), as well as in lung adenocarcinoma DNA from never-smokers (23). Reduced MGMT protein expression has been thought to result in a spontaneous ‘mutator’ phenotype; however, an analysis of spontaneous in vivo lacI mutations in MGMT-deficient (Mgmt–/–) mice has failed to support this notion (24). As Mgmt–/– mice are more prone to tumorigenesis and cytotoxicity following alkylating agent exposure (17), reductions in the expression of this repair molecule might promote neoplasia given the presence of either endogenous or exogenous sources of alkylation stress.

We thus examined the ability of MGMT to prevent mutations in two tissues exposed to an NNK challenge. To achieve this, lacI reporter gene (25) mutant frequencies (Mf’s) and mutational spectra of Mgmt-deficient lung and liver, primary sites of NNK metabolism, were determined. NNK has previously been shown to produce Mf inductions in both the lacZ and cII gene reporters of wild-type (WT) MutaMouse (26–28). Employing the lacI-based Big Blue reporter system, we demonstrated that NNK treatment of mice induced a complex spectrum of mutations in lung and liver, and that in Mgmt–/– hosts it led to a further increase in the numbers of G:C to A:T transitions. The latter were accompanied by a shift in their positions from CpG to GpG sites, consistent with the activities of SN1 alkylating agents, such as methylnitrosourea (29) and specific NNK metabolites.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Transgenic lines and genotyping procedures
Mgmt-deficient mice (30), generously provided by L. Samson (Massachusetts Institute of Technology) were crossed with Big BlueTM mice (Stratagene, La Jolla, CA) to generate Mgmt–/–lacI+ animals, all on a C57BL/6 background. For controls, WT littermates were used. Mice were housed in microisolator-type cages at 25°C with a 12 h light/dark cycle in a viral antibody-free barrier facility in accordance with University of Calgary Animal Care Committee and Canadian Council of Animal Care guidelines. Irradiated, pelleted Mouse Diet (Purina Picolab #5058) and water were given ad libitum. Mgmt and lacI genotyping were carried out by polymerase chain reaction using DNA extracted from mouse tail tips as described previously (24).

Chemicals and NNK treatment of mice
O6-[1,2,2-2H3-4-oxo-4-(3-pyridyl)butyl]-2'-deoxyguanosine ([1,2,2-2H3]-O6-pobdG), O6-[2H3]-methyl-2'-deoxyguanosine ([2H3]O6-mdG) and [13C12H3]-7-methyl-2'-deoxyguanosine were synthesized according to published methods (31–33). [13C12H3]-7-methylguanine ([13C12H3]-7-mG) was prepared by heating the 2'-deoxyguanosine derivative under acidic conditions at 80°C for 30 min. NNK (Toronto Research Chemicals, Toronto, ON) was dissolved in 100% dimethyl sulfoxide (Sigma-Aldrich, St Louis, MO) as recommended by the NNK supplier to ~1070 µmol/ml and single dose aliquots stored at –80°C for a maximum of 3 weeks. On injection days, further dilutions to 25 µmol/ml were done in 0.9% saline (Baxter, Deerfield, IL). NNK (25 µmol) was given via the intraperitoneal route to 7- to 8-week-old mice (with the exception of one mouse that was treated at 14 weeks) once a week for three consecutive weeks (for a total of 75 µmol NNK), followed by a 1-week period to allow for mutation fixation, prior to euthanasia. The NNK dose selected was ~250 mg/kg/injection, as described by Hashimoto et al. (26), and based on the average weight of ~7-week-old male C57BL/6 mice in our colony. In a pilot test, dimethyl sulfoxide diluted in saline was injected in the manner described above to determine if there were induced mutations; furthermore, frozen aliquots were also compared with fresh dilutions of NNK; however, no difference in mutation frequency was noted in either case (data not shown). For the short-term adduct analysis, mice lacking MGMT and WT controls were given a single NNK (250 mg/kg) dose intraperitoneally and 24 h later lungs and livers were harvested from the mice for DNA preparation.

Tissue extraction and genomic DNA isolation
Following euthanasia by CO2 inhalation, lung, liver and pancreas were rapidly removed, flash-frozen in liquid nitrogen and stored at –80°C. For the lacI studies, tissue DNA isolation was carried out using RecoverEaseTM (Stratagene) according to the manufacturer's instructions. Further purification of DNA samples in preparation for adduct analysis by mass spectroscopy was carried out as follows: The RecoverEaseTM (Stratagene) protocol was used according to the manufacturer's instructions until the isolation of the nuclear pellet step after which a NucleoBond Nucleic Acid Purification Kit (Clontech) was used. In brief, the pellet was re-suspended and proteinase K digestion performed as per the manufacturer's instructions. The digested mixture was added to an equilibrated NucleoBond AX-G500 column (Clontech) and allowed to enter the column by gravity flow. The column was washed and 1 ml fractions were collected. Spectrophotometer readings were used to determine the A260/A230 and A260/A280 ratios of the fractions. Fractions with readings of A260/A230 > 2.0 and A260/A280 > 1.8 were combined and the DNA was precipitated with 0.7 volumes of isopropanol and transferred to a glass test tube. The DNA was washed with 4x 1 ml 70% ethanol followed by 4x 1 ml 100% ethanol and dried under a stream of nitrogen gas.

Determination of lacI Mf‘s and mutation spectra
The transgenic {lambda}-phage rescue procedure was carried out as described in the Big BlueTM protocol (Stratagene). Mf was calculated by determining the ratio of mutant (blue) to WT (clear) plaques. For each DNA sample recovered from various tissues, a minimum of 150 000 plaques were enumerated. Blue plaques were confirmed by replating (24), and randomly selected mutants were then subjected to sequence analysis. To assemble mutation spectra, the complete (~1 kb) lacI sequence of 8–18 unique mutants per tissue sample was determined by the DNA Sequencing Core Facility (University of Victoria, British Columbia, Canada) using primers starting at lacI gene positions –234 and 1337.

Data analysis and databases
Two lacI mutation databases were used to obtain data for comparative analysis: (i) the database for mutations and Mf‘s in Big BlueTM, http://eden.ceh.uvic.ca/results.htm, now offline, was kindly accessed for us by Dr J. de Boer (University of Victoria, Victoria, Canada) and (ii) the Transgenic-Bacterial lacI database and MutaBase Software were downloaded from http://www.ibiblio.org/dnam/des_laci.htm (34). Upon analysis of the mutation spectra, clonal mutations (mutations occurring more than once in a given tissue sample, potentially arising from a common progenitor) were excluded from the analysis; however, recurrent lacI mutations (i.e. ‘hotspots’), defined as those observed in the same tissue from more than one animal, were retained. A correction was also applied for lacI mutations originating in bacteria (mutations that potentially occurred ex vivo). Hallmark bacterial mutations were identified through various databases and publications (34–36). To determine the effect of correcting for putative clonal and bacterial mutations, spectra with and without correction were compared via the Monte Carlo estimation of the P-value of the hypergeometric test (MCA) (34,37). No significant differences were evident (data not shown). MCA was used to make pairwise statistical comparisons of up to 12 mutant categories from the various genotypes (34,37). The program was run with 1700 iterations and significance was set at {alpha} = 0.05. Student's t-tests were performed using Microsoft Excel.

DNA adduct analysis following NNK exposure
DNA (50–100 µg) was dissolved in 10 mM sodium phosphate, pH 7 (200 µl), and spiked with [13C12H3]-7-mG (5–25 pmol). The solution was heated at 95°C for 30 min. After cooling on ice for 30 min, 1/10 volume of 1 N HCl was added to precipitate the DNA. Following centrifugation, the supernatant was reserved for 7-mG analysis. A 0.1 N HCl solution (500 µl) was added to the pellet followed by [2H3]O6-mdG (0.5–5 pmol) and [1,2,2-2H3]-O6-pobdG (0.2–2 pmol). This mixture was heated at 80°C for 30 min. A portion of the hydrolysates (50 µl) was reserved for the determination of guanine concentration. The remainder of the acid hydrolysate was applied to a Strata-X C18 cartridge (30 µm, 30 mg) (Phenomenex, Torrance, CA). The cartridge was sequentially eluted with 1 ml water, 1 ml 10% methanol and 1 ml 100% methanol. The adducts eluted in the 100% methanol fraction was collected and concentrated to dryness under reduced pressure. The residue was re-suspended in 25 mM ammonium acetate (30 µl) for LC/MS/MS analysis.

Capillary LC/ESI–MS/MS analyses were carried out on a TSQ Quantum Ultra AM mass spectrometer (Thermo Electron, Bellafonte, PA) interfaced with an Agilent 1100 series capillary High Pressure Liquid Chromatography (HPLC) (Agilent Technologies, Palo Alto, CA). They were performed in the positive ion mode with N2 as the nebulizing and drying gas. The voltage setting was 3.8 kV and the heated capillary was set at 250°C. For the analysis of 7-mG and O6-mG, samples (injection vol: 8 µl) were separated on a 250 x 0.5 mm, 4 µm Synergi C18 column (Phenomenex, Torrance, CA). For 7-mG analysis, the column was eluted with an 8 min linear gradient from 25 mM ammonium acetate with 9% of methanol containing 25% acetonitrile to 25 mM ammonium acetate with 18% methanol containing 25% acetonitrile at a flow rate of 13 µl/min. For O6-mG, the column was isocratically eluted with 25 mM ammonium acetate with 18% of methanol containing 25% acetonitrile at a flow rate of 12 µl/min. For O6-pobG analysis, acid hydrolysates were separated on a Zorbax SB-C18, capillary column (150 x 0.5mm, 5 µm) (Agilent Technologies, Santa Clara, CA) with a 20 min linear gradient from 15 mM ammonium acetate containing 10% acetonitrile to 15 mM ammonium acetate containing 30% acetonitrile followed by a 2 min gradient to 15 mM ammonium acetate containing 50% acetonitrile and held for 4 min at a flow rate of 10 µl/min. Quantification was done with selected reaction monitoring. The mass transitions (parent to product) monitored were 166->149 for 7-mG and O6-mG, 169->152 for [2H3]-O6-mG, 170->153 for [13C12H3]-7-mG, 299->148 for O6-pobG and 302->151 for [2H3]-O6-pobG. The amount of 7-mG, O6-mG and O6-pobG in each sample was determined from the product of the area ratio (O6-pobG/[2H3]O6-pobG) and the amount of internal standard added. Calibration curves were constructed from known amounts of authentic standards spiked with known amounts of internal standard. Guanine concentrations were determined as described previously (38). The amount of DNA adducts were normalized to the amount of the guanine in each sample.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Mgmt deficiency led to a trend of increased lacI Mf in NNK-treated lungs and livers
To evaluate the ability of MGMT to protect a chromosomally integrated transgene locus from NNK-induced mutations, lacI Mf were determined for three NNK target organs isolated from Mgmt–/– and WT Big BlueTM transgenic mice. Lung was selected because NNK is a potent lung carcinogen in laboratory animals and has been shown to cause mutations in this tissue (3,4,9,26). The liver was chosen since MGMT activity levels are highest in this organ (17,39) and mutational inductions have been reported for this tissue following NNK administration to mice (26). Finally, the pancreas was analyzed because NNK has been shown to induce pancreatic tumors in rats (4,40) and also because baseline lacI mutant frequencies for pancreas have yet to be reported.

The lung contains P450 oxidoreductases capable of metabolizing NNK, yielding reaction products that form potentially mutagenic DNA adducts (2); consequently, an increase in lacI Mf was predicted. Indeed, following NNK exposure, WT lung DNA exhibited a ~5-fold (P = 0.01) increase in lacI Mf compared with untreated controls (Table I), demonstrating that the transgenic lacI reporter system was able to detect NNK-induced mutations in vivo. Even though we compared Mf from 11- to 12-week-old treated mice to historical data derived from 7-week-old untreated WT mice, we would expect the Mf of the latter to be similar to that of untreated 11- to 12-week-old mice (41). Mgmt-deficient lungs exposed to NNK demonstrated a further increase in Mf (19.5 x 10–5) compared with WT-treated lungs (14.6 x 10–5) (Table I); however, this increase did not reach significance (P = 0.27). The baseline lung DNA Mf was anticipated to be equivalent for WT and Mgmt–/– mice (Table I) given that the spontaneous Mf of two Mgmt–/– tissues (small intestine and liver) was previously been shown to be similar to that of WT tissues (24). The results obtained from NNK treatment of Mgmt–/– mice therefore suggested that the levels of NNK-derived O6-dG lesions left uncorrected as a result of Mgmt deficiency were insufficient to significantly alter the lacI Mf in lung DNA.


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  		Table I. lacI mutant frequencies for DNA obtained from lung of Mgmt-deficient and WT Big BlueTM mice treated with NNK

 
Mouse liver was previously reported to show higher DNA adduct levels and Mf than lung, following NNK exposure (4,26). Since MGMT levels and activity are the highest in liver (17), we anticipated that MGMT deficiency in this organ would result in greater NNK-induced increases in Mf than were seen in the lung. Starting from baseline liver lacI Mf that were similar between WT and Mgmt–/– mice, we observed strong Mf inductions in response to NNK administration to both WT (3-fold over WT; P = 0.01 and 4-fold over Mgmt–/–; P = 0.01, respectively) and Mgmt–/– mice (7-fold over WT; P = 0.04 and 11-fold over Mgmt–/–; P = 0.04, respectively) (Table II). There was a trend for an increase in Mf in the NNK-treated Mgmt–/– livers (26.9 x 10–5), as compared with the treated WT control livers (10.4 x 10–5). However, the variability was relatively high, so that the observed ~3-fold increase in Mf in the NNK-treated Mgmt–/– mice did not reach statistical significance (P = 0.11) (Table II). Also, in contrast to the findings of Hashimoto et al. (26), we found that the Mf of treated livers and lungs were similar. As observed in the lungs, the results suggested that NNK-induced O6-dG adducts were not sufficiently high enough in Mgmt-deficient livers, as compared with WT, to significantly impact the Mf. Despite this, the livers exhibited a much stronger trend towards increases in Mf (Table II) when compared with the lung Mf (Table I).


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  		Table II. lacI mutant frequencies for DNA obtained from liver of Mgmt-deficient and WT Big BlueTM mice treated with NNK

 
To our knowledge, the pancreas has not previously been analyzed by in vivo mutational reporter transgenes. We successfully extracted and plated DNA from the pancreas; however, our preliminary data revealed no clear induction in the Mf following NNK exposure. The baseline Mf was within the range reported for all Big BlueTM tissues examined, namely 1.4–6.7 x 10–5 (41–44). No difference was apparent between NNK-treated WT (2.0 x 10–5; N = 1) or Mgmt-deficient (3.23 x 10–5; N = 4) mice (data not shown). Consistent with this result, a study of human pancreatic DNA from tobacco cigarette smokers also found no increase in 4-hydroxy-1-(3-pyridyl)-1-butanone, a metabolic product indicative of DNA damage (45). However, evidence of NNK metabolism by alpha-hydroxylation in human pancreatic tissue has been reported (45). The lack of induction in Mf in murine pancreas might be due to relatively low expression levels of the specific cytochrome P450 oxidoreductases required for NNK metabolism. In summary, NNK administration led to obvious lacI mutational inductions in the lungs and livers of WT mice, and deficiency of Mgmt produced a trend for a further increase in Mf. This trend was most evident in the livers of NNK-treated Mgmt–/– mice (P = 0.1).

Although the DNA adduct study (see below) revealed the persistence of O6-MeG, particularly in liver DNA, we did not see a significant increase in the numbers of sectored plaques. The latter would have been indicative of mutations generated during the bacterial phase of the Big Blue assay as a result of adducts present on the incoming phage DNA. However, as bacteria would be infected on average by only one phage genome, the bacterial alkyltransferase would be able to readily purge the DNA molecule of any residual O6 alkylations.

Mgmt deficiency alters the mutational spectrum of NNK-treated lung DNA
Although no significant differences in the Mf of NNK-exposed Mgmt-deficient and WT mice were seen, it was still important to determine whether qualitative changes were present. An analysis of the lacI mutational spectrum can reveal specific mutagenic events that are not evident from analyses of Mf data alone. Since DNA replication across alkylated O6-alkyl dG residues can result in G:C to A:T transitions, the frequency of this type of mutation was predicted to be increased in the absence of MGMT. Thus, we examined whether lack of MGMT in lung and liver was associated with a specific change in the NNK-induced mutational spectra.

The mutational spectra of lacI genes rescued from lung DNA in both WT, and WT NNK-treated mice (Table III), revealed a predominance of G:C to A:T transitions, 61% and 44%, respectively. However, unlike WT-untreated mice where the second most common class of mutation was the G:C to T:A transversions (10%), the A:T to T:A transversions (19%) were the next most abundant class in WT NNK-treated mice, followed by A:T to C:G transversions (11%) (Table III). The induction in transversions resembled that reported by Hashimoto et al. (26) in lung and liver DNA from NNK-treated mice. We found that NNK treatment of Mgmt-deficient lungs led to a modest increase in G:C to A:T transitions (57%), as compared with NNK-treated WT lungs (44%) (Table III). However, the percent change in neither the full spectra (P = 0.12) nor the transitions and transversions alone (P = 0.19) were significantly different between the two groups.


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  		Table III. lacI mutation spectra of Mgmt–/– lung DNA from 7- to 8-week-old mice treated with NNK compared with the lacI mutation spectra of Big BlueTM control mice

 
Given the Mf induction in the lungs of the NNK-treated animals, we considered the absolute numbers of specific mutations (i.e. multiplying mutational percentages by the average Mf) as this would give a better idea as to the frequency of each mutational class (48,49). By this analysis, there was increase in G:C to A:T mutations from 1.7 x 10–5 in untreated mice to 6.5 x 10–5 in NNK-treated WT mice and to 11.0 x 10–5 in NNK-treated Mgmt–/– mice (Table III). Thus, although not obvious in terms of the percent change, the absolute numbers revealed an increase in G:C to A:T transition mutations within the NNK-treated groups. The 1.3-fold increase in Mf between NNK-treated WT and Mgmt–/– lungs results primarily from an increase in the number of deletions and transitions, whereas the absolute number of transversions remained unaltered by Mgmt deficiency (Table III).

Following NNK, there was a shift in the positions of a proportion of the G:C to A:T transitions from CpG to GpG sites. This was particularly prominent in Mgmt-deficient lungs, where 49% of these transitions were at GpG sites (Table III), up from the 25% observed for the WT lungs. G:C to A:T transition mutations at CpG sites can result from spontaneous deaminations of 5-methylcytosine residues (producing a G:T mismatch), whereas the second dG of GpG sites tends to be a hotspot for alkylating agents such as methylnitrosourea (29). Together, these results were consistent with NNK metabolite-derived alkylations of the O6 position of dG in lung DNA and suggested that MGMT was important for the repair of these lesions.

Mgmt deficiency alters the lacI mutational spectrum of NNK-treated liver DNA
The lacI mutation spectra of NNK-treated livers generally followed the same trends observed in the lungs (Tables III and IV). The absolute number of transversion mutations was higher in WT NNK-treated livers than in WT-untreated livers, but NNK-induced transversions were similar between WT and Mgmt–/– livers (Table IV). In addition, deletions were also more frequent in WT liver DNA (3.1 x 10–5) and Mgmt-deficient (2.9 x 10–5) NNK-treated mice, than in the untreated controls (0.3 x 10–5) (Table IV). Importantly, and consistent with a lack of MGMT activity, a striking induction in transitions was observed in response to NNK when WT (30% G:C to A:T), and Mgmt–/– (66% G:C to A:T) liver DNAs were compared (Table IV); this represented an ~6-fold increase in the absolute number of these mutations in the Mgmt–/– livers (Table IV). Pairwise comparisons of the percent change in the complete lacI mutation spectra using the stringent MCA test revealed highly significant differences (P < 0.000) between NNK-treated WT and Mgmt–/– mice, and this held even when the transition and transversion categories were compared (P = 0.001). MCA of all the lacI spectra in the liver revealed significant differences except when the untreated WT and Mgmt–/– were compared; the latter observation was consistent with the findings of Sandercock et al. (24). As observed in the Mgmt–/– lung DNA, a dramatic shift in the proportion of G:C to A:T transitions from CpG to GpG sites was again evident when the NNK-treated Mgmt–/– and WT livers were compared; this amounted to 40% and 13%, respectively. Taken together, the observed increase in transition mutations and shift in these mutations towards GpG sites in NNK-exposed Mgmt-deficient mice was consistent with MGMT being required for the repair of the O6-dG lesions induced by this carcinogen.


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  		Table IV. lacI mutation spectra of Mgmt–/– liver DNA from 7- to 8-week-old mice treated with NNK compared with the lacI mutation spectra of Big BlueTM control mice

 
The effects of MGMT deficiency on the levels of specific NNK metabolite-derived adducts was more striking in liver than in lung DNA
Analysis of DNA adduct levels 1 week after the final treatment of NNK indicated that the levels of 7-mG in the liver (Mgmt–/–: 127 ± 19 pmol 7-mG/µmol guanine; Mgmt+/+: 151 ± 2 pmol 7-mG/µmol guanine) and the lung (Mgmt–/–: 45 ± 11 pmol 7-mG/µmol guanine; Mgmt+/+: 37 ± 10 pmol 7-mG/µmol guanine) were similar between WT and Mgmt-deficient mice, indicating that there were no significant differences in NNK metabolism between the two strains of mice. However, there were substantial differences in the levels of O6-mG detected in the livers of NNK-exposed MGMT-deficient mice compared with WT mice (Table V). Indeed, adduct levels in liver DNA of mice expressing MGMT were at least an order of magnitude lower than those of Mgmt–/– livers. This difference increased in animals that received multiple treatments of NNK. A similar trend was also observed for O6-pobG. These data are consistent with previous observations showing that O6-mG and O6-pobG are efficiently repaired by MGMT in the livers of NNK-treated A/J mice (14,50).


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  		Table V. Levels of O6-mG and O6-pobG adducts in NNK-treated micea

 
There were smaller differences between the levels of O6-mG and O6-pobG in the lung DNA between Mgmt-deficient and WT mice. These adducts tend to be more persistent in lung than in liver DNA, in part, because the levels of MGMT in the lungs is ~10-fold less than the level in the livers of WT mice (14). MGMT levels can also be suppressed for some time following exposure to NNK (14). Since NNK generates adducts that deplete MGMT activity and the levels of this molecule are known to be low in the lung, the data shown in Table 5 support the hypothesis that the lung is less well equipped to deal with O6-alkylguanine DNA damage. Some repair still occurs in the lung, however, since the levels of O6-mG and O6-pobG are lower in the WT mice as compared with the Mgmt-deficient mice. The relative levels of O6-mG and O6-pobG shown in Table V were greater than or equal to those reported for A/J mice treated with a carcinogenic dose of NNK (14,50).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
NNK, a potent derivative of the tobacco carcinogen, leads to a variety of DNA adducts, and in particular O6-mG, whose levels have been shown to correlate directly with lung tumor incidence in rodents (4). Left unrepaired, O6-mG leads to the formation of G:C to A:T transitions that have the potential to promote both oncogene activation and tumor suppressor gene dysfunction (51). O6-mG is not the only DNA lesion caused by metabolites of NNK, for example, DNA pyridyloxobutylation can also generate mutagenic adducts, resulting in transitions and transversions (10). The model pyridyloxobutylating agent, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone induced transitions and transversion mutations in the Ki-ras gene in A/J mice (9), and the MGMT substrate, O6-pobG, produced both G:C to A:T and G:C to T:A mutations in human cells (10). In addition, other pyridyloxobutyl DNA adducts that are not MGMT substrates may also be mutagenic (52–54). The specific mutagenic properties of the latter have not been investigated. Lastly, mutations could occur as a result of NNK metabolism-associated generation of reactive oxygen species. The latter are known to produce various DNA lesions, including 8-oxoG residues and single-strand breaks (7). As discussed below, the diversity of the known and potential mutagenic effects of NNK metabolites was reflected by the relatively complex spectrum of lacI mutations recovered from mice exposed to this agent.

Since O6-mG and O6-pobG are both substrates for the DNA repair enzyme MGMT (12,50,53), it suggested that this ‘suicide enzyme’ repair pathway provides protection against mutagenesis associated with ingestion of tobacco product-derived nitrosamines. Herein, we tested this hypothesis by exposing Mgmt–/– and WT control mice containing the Big BlueTM in vivo mutational reporter system to NNK. These crosses enabled us to evaluate how MGMT regulated the mutational responses to this carcinogen. The Big Blue transgenic line has been found to be a useful tool for studying in vivo occurrence of point mutations, frameshifts and small insertion/deletions in any tissue (25,29). Although the lacI mutational target is a prokaryotic gene sequence, it has been found that the mutational spectra of the lacI gene and endogenous loci (typically Hprt) following mutagen exposures are highly congruent (55–59). Given its high sensitivity to point changes, the lacI reporter was ideally suited for the detection of NNK-induced mutations.

The NNK dosage used in this study was primarily based on the success of a previous study showing mutational inductions in another reporter strain (26). However, the dose in our study (75 µmol = 15.5 mg or 775 mg/kg assuming an average mouse weight of 20 g) is ~700 times higher than the average lifetime NNK dose in someone who has smoked for 40 years (~410 µmol = 85 mg or 1.1 mg/kg assuming an average human weight of 75 kg). The human lifetime consumption is similar to the lowest total NNK dose shown to induce lung tumors in rats (1.8 mg/kg) (4), but in order to study both the acute mutagenic effects of NNK in vivo and the NNK metabolite-derived DNA adduct levels in tissues, relatively high doses of this chemical had to be used. The doses of NNK that we employed resulted not only in the generation of NNK metabolite-derived DNA adducts in lung and liver but also yielded clear lacI Mf inductions in these two tissues. From the Mf data (Tables I and II), it is evident that lower doses of NNK would likely yield inconclusive data. Our results were in line with previous in vivo studies examining adduct levels in the lungs and livers of NNK-treated A/J mice (4) and reporter gene Mf inductions in MutaMouse (CD2 hybrid mouse) (26). As in previous studies, NNK-induced methyl DNA adduct levels were higher in liver than in lung DNA (Table V). Although we measured only one pyridyloxobutyl DNA adduct, O6-pobG, this species was present at comparable levels in lung and liver DNA at 24 h post-NNK exposure.

We observed a trend towards higher Mf following NNK treatment of mice lacking MGMT as compared with WT controls. This increase was higher in Mgmt–/– livers (26.9 x 10–5) than the Mgmt–/– lungs (19.5 x 10–5) (Tables I and II). The lack of a greater difference between WT and knockout mice was surprising given the reports of increased Mf and/or increased sensitivity of Mgmt–/– mice to alkylating agents (18,30,60–62), including temozolomide (18,30), streptozotocin (30) or dacarbazine (61), and the diminished sensitivity of mice over-expressing MGMT to such chemicals (63–68). However, it should be noted that the various metabolites of NNK generate a complex spectrum of DNA adducts, several of which are not only potentially mutagenic but are also unlikely to be candidates for MGMT-mediated repair.

The Mf increases that could be attributed to MGMT deficiency were higher in the liver than in the lung. This can be explained, at least in part, to differences in MGMT expression between these tissues. Following NNK treatment, adduct formation was similar between WT and Mgmt–/– mice as indicated by the similar levels of 7-mG in both groups, with the lungs experiencing one-third of the amount of DNA damage as the liver. However, compared with lung, O6-mG and O6-pobG levels in liver DNA were dramatically higher in Mgmt–/– mice, in part because these adducts are rapidly reduced due to the high levels of MGMT in the WT liver (Table V). This would also result in attenuation of G:C to A:T transitions in the mutational spectrum of the WT liver. In contrast, knockout of Mgmt in the lungs exerted only a modest impact on the levels of O6-mG and O6-pobG adducts, and the Mf difference between WT and Mgmt–/– lungs was diminished. This was likely because lung MGMT levels are ~10 times lower than those of the liver (14). In addition, previous studies have shown that MGMT levels in liver are restored more rapidly than in lung following a carcinogenic dose of NNK. For example, MGMT levels in A/J mice lung were suppressed for >4 days following a single dose of NNK (14). Therefore, NNK administration might, in effect, be generating a virtual MGMT ‘knockout’ in the lungs, but not the livers of the WT mice. Together, these factors predicted that there would be higher levels of O6-mG and O6-pobG DNA in Mgmt–/– liver than in lung. Indeed, when the levels of the two adduct types were compared, there was ~3-fold higher O6-mG and 2-fold higher O6-pobG in the MGMT-deficient livers relative to the lungs (Table V). Consequently, the increase in lacI Mf was less in the lungs than in the livers of the Mgmt–/– mice, paralleling the differences in levels of O6-alkylguanine adducts. In summary, our DNA adduct study provides a rationale for differential mutational effects of NNK observed in Mgmt–/– liver versus lung DNA samples.

As mentioned in the first paragraph of the discussion, other types of pyridyloxobutyl DNA adducts, which do not represent MGMT substrates (53), may have been contributing to the observed Mf and thus serving to dilute-out the contributions to the Mf that were due specifically to the loss of MGMT. Another reason for the Mf not being more significantly elevated in the Mgmt–/– hosts after NNK exposure is that other DNA repair systems such as base excision repair and DNA mismatch repair, whose activity might also be differentially regulated between lung and liver, may have been recruited to limit the mutagenic effects of NNK-derived adducts that would ordinarily be processed by MGMT. Attesting to the importance of mismatch repair, loss of this repair system can greatly increase the levels of G:C to A:T transitions in the lacI system following treatment with an SN1 alkylating agent (29), an effect that would be even more marked if O6-mG adducts were allowed to persist in cellular DNA owing to a lack of MGMT (17,69). In this vein, another possible factor limiting the level of the lacI Mf inductions in Mgmt–/– mice would be the increased susceptibility of MGMT-deficient, mismatch repair proficient cells to DNA alkylation-induced apoptosis (69). Thus, Mgmt–/– cells sustaining the highest levels of NNK metabolite-induced O6-mG adduct formation (and hence exhibiting the highest lacI mutation levels) might undergo apoptosis, eliminating their contribution to the Mf.

Even in the absence of statistically significant changes in the Mf (which generally result from inter-animal variability), important information can still be gleaned from analyses of mutational spectra. The transition mutation class was predicted to be most affected by Mgmt deficiency, since unrepaired O6-dG alkylations result primarily in G:C to A:T changes during DNA replication. However, consistent with the findings of Hashimoto et al. (26), we also found that NNK led to increased transversions in the lung, particularly A:T to T:A and A:T to C:G changes. Although the DNA adducts responsible for A:T to C:G mutations are not well defined, those responsible for A:T to T:A changes were proposed to be due to N3-alkyl and O2-alkylthymine (26,70). The relative contributions of the pyridyloxobutylation and methylation pathways likely responsible for these different classes of mutations could potentially be separated experimentally through the treatment of the mice with specific NNK analogues that generate only pyridyloxobutyl adducts or methyl adducts of DNA bases. Thus, for example, to study pyridyloxobutylation-induced mutations, Big BlueTM mice could be treated with 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (9,71).

In contrast to the lung, livers revealed a significant difference in their NNK-induced mutational spectra when the lacI mutational profiles of WT and Mgmt–/– samples were compared. In the WT liver, G:C to A:T transition levels remained low, likely a result of the high MGMT expression levels found in this tissue. The latter possibly provide an explanation for the relative rarity of NNK-induced liver tumors in WT mice. In contrast, in the absence of MGMT, NNK-exposed livers exhibited significant increases in G:C to A:T transitions when compared with WT livers (6-fold) and lungs (2-fold) (Tables III and IV). The change in the positions of a large fraction of G:C to A:T transitions from CpG to GpG sites (Table III) in Mgmt–/– lung and liver DNA samples was striking. Since GpG sites are targets for adduct-forming SN1-type metabolites [e.g. methylnitrosourea (29)], the increase in G:C to A:T transitions at these sites indicated a role for MGMT in the repair of NNK-induced adducts at these sites. The data showing high levels O6-mG adducts in the DNA samples supported these findings.

Interestingly, an increase in deletion mutations following NNK treatment was apparent within the lacI mutational spectra of both the lung and liver. Mgmt–/– lungs demonstrated increased deletions over WT, whereas the opposite was true of liver. Hashimoto et al. (26) also observed an increase in deletions within the cII spectra of NNK-treated mouse lung and liver DNA. As lacI gene deletion mutations commonly occur within bacteria, we took care to identify and remove from the analysis any of the hallmark bacterial lacI mutations, as these could have occurred ex vivo in the bacterial host (see Materials and Methods). The mechanisms responsible for the deletions observed in our mice are not clear. However, since NNK may lead to single-strand DNA breaks, it is possible that such lesions would engender slippage reactions or double-strand breaks whose repair could result in deletions (46,72). In addition, site-specific mutagenesis studies have indicated that O6-pobG can cause deletion mutations (10). Another intriguing possibility is that DNA adducts could be leading to deletions via a two-step process of misinsertion opposite base adducts, followed by misalignment with adjacent or remote complementary sequences. If a bypass DNA polymerase was involved in the repair of NNK lesions, this might also lead to deletions (73). Finally, the difference in spectra between the lung and liver could have been influenced by qualitative differences how NNK was metabolized. For example, differences in ratios of P450 oxidoreductase isoforms between liver and lung can result in the formation of distinct metabolites, for instance, the formation of ketoalcohols, such as 4-hydroxy-1-(3-pyridyl)-1-butanone that is limited to the liver (74).

Extrapolating to humans from our previous studies of Mgmt–/– mice (24), it is possible that bronchial epithelial cells with diminished or reduced MGMT would not show a spontaneous increase in mutant frequency. Instead, our results suggest that a change in mutation spectrum with the trend towards an increase in mutation frequency would be observed in MGMT-deficient cells exposed to specific carcinogens such as the tobacco nitrosamine, NNK. Indeed, the generation of G:C to A:T transition mutations is likely key to NNK-induced carcinogenesis, since this is the predominant mutation found in the K-ras genes of NNK-induced lung tumors in rodents (4). Previous studies have also shown that G:C to A:T transitions are carcinogenic (4,51). Supporting this, Liu et al. (66) showed a reduction in both tumor number and K-ras mutations following transgenic expression of hMGMT in NNK-exposed A/J mice. Therefore, MGMT promoter methylation (seen in individuals with non-small cell lung cancer and also in the pre-cancerous states seen in some tobacco smokers) (19–22) may indeed represent a marker for lung cancer susceptibility. In addition, even without MGMT promoter methylation, tobacco product smoking and the NNK thus ingested could in itself potentially result in chronic MGMT depletion in the lung, thus augmenting the mutational responses of bronchial cells to this chemical.

We have provided evidence for the importance of MGMT in the repair of NNK-induced DNA lesions in vivo. Tumorigenesis studies of Mgmt-deficient mice exposed to NNK would be needed to confirm the suspected link between MGMT, O6-mG adducts and lung tumors, although studies of A/J mice, where the total number of O6-mG adducts correlated directly with the number of NNK-induced lung tumors (4), suggest that MGMT deficiency in this strain would greatly accelerate tumorigenesis. Lack of MGMT might also lead to NNK-induced neoplasms in other organs, notably the liver, a site where we observed a dramatic increase in level of G:C to A:T transitions. Furthermore, mutagenesis and tumorigenesis studies comparing NNK metabolites such as the pyridyloxobutylating agent, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone, and the methylating agent, N-nitroso(acetoxymethyl)methylamine, would provide an indication not only about the genesis of particular mutations but also a better understanding of the nature of the adducting species required for tumor induction. In addition to tobacco nitrosamines, the Mgmt–/– mouse model would be useful for in vivo studies of other nitrosamines, such as those found in certain Asian foodstuffs, or in well water (such as N-nitrosodimethylamine), that are thought to pose carcinogenic risks (75).


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
National Cancer Institute of Canada with funds from the Canadian Cancer Society (to F.R.J.), and also by grant R01-CA115309 from the National Cancer Institute (to L.A.P.). Alberta Heritage Foundation for Medical Research post-doctoral fellowships (to L.E.S. and H.A.L.).


   Acknowledgments
 
We are indebted to Dr L. Samson for providing us with the Mgmt-deficient mouse strain, to M. Villemaire and L. Forster for their excellent technical assistance and to L. Zimmerman for maintenance of the animal colony. J.N.H. held a Natural Sciences and Engineering Research Council studentship, and F.R.J. was the recipient of a Canada Research Chair award.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received March 21, 2007; revised December 28, 2007; accepted January 24, 2008.


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