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Carcinogenesis Advance Access originally published online on March 6, 2007
Carcinogenesis 2007 28(8):1824-1830; doi:10.1093/carcin/bgm051
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Inhibition of vinyl carbamate-induced mutagenicity and clastogenicity by the garlic constituent diallyl sulfone in F1 (Big Blue® x A/J) transgenic mice

Lya G. Hernandez and Poh-Gek Forkert*

Department of Anatomy and Cell Biology, Queen's University, Kingston, Ontario K7L 3N6, Canada

* To whom correspondence should be addressed. Tel: +1 613 533 2854; Fax: +1 613 533 2566; Email: forkertp{at}queensu.ca


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Vinyl carbamate (VC) is a metabolite of ethyl carbamate (EC), a naturally occurring compound found in fermented foods and alcoholic beverages. CYP2E1 mediates the sequential oxidation of EC to VC and subsequently to the vinyl carbamate epoxide, which is believed to be the ultimate carcinogenic species. Here, we have tested the hypothesis that bioactivation of VC by CYP2E1 plays a central role in the development of its mutagenicity and clastogenicity, and further that inhibition of CYP2E1 by diallyl sulfone (DASO2) leads to diminution in their incidences. DASO2 is a garlic constituent that is oxidized by CYP2E1, leading to inactivation of this P450. F1 (Big Blue® x A/J) transgenic mice harboring the {lambda} cII gene were used for in vivo identification and quantitation of mutations in the lung and small intestine. Mice were pre-treated with DASO2 (12.5–200 mg/kg, p.o.), treated 2 h later with VC (60 mg/kg, i.p.) and were killed 4 weeks later. Our results showed that pre-treatment of mice with DASO2 at doses of 50–200 mg/kg significantly decreased the VC-induced mutant frequencies (MFs) by 50–70%. In the small intestine, pre-treatment with 200 mg/kg of DASO2 decreased the MF by ~40%. Clastogenicity, as assessed by the frequency of micronucleated reticulocytes, was significantly decreased (33–44%) by pre-treatment with DASO2 (50–200 mg/kg). These results demonstrated that bioactivation of VC by CYP2E1 plays a valid role in the development of mutagenicity and clastogenicity, and further that inhibition of this pathway by DASO2 produces a protective effect.

Abbreviations: DASO2, diallyl sulfone; {epsilon}dAS, 1,N6-ethenodeoxyadenosine; EC, ethyl carbamate; EDTA, ethylenediaminetetraacetic acid; MF, mutant frequency; PCR, polymerase chain reaction; VC, vinyl carbamate; VCO, vinyl carbamate epoxide


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Vinyl carbamate (VC) is derived from oxidative metabolism of ethyl carbamate (EC), a compound used historically as a cosolvent with analgesic and sedative drugs (1), as well as an anti-neoplastic agent for treatment of chronic leukemia and multiple myeloma (2). EC is formed in fermentation of alcoholic beverages and food products including bread, cheese, yogurt, soy sauce and vinegar (3–5). The amounts of EC found in various food items included the following: stone-fruit brandies (2000 ng/g), table wines (10–15 ng/g), sake rice wine (130 ng/g), bread (7 ng/g), yogurt (<1 ng/g) and soy sauce (18 ng/g) (5). Hence, human exposure occurs inadvertently via consumption of alcoholic beverages and fermented foods. The mean daily intake of EC from various food items in adults has been estimated to be ~10–20 ng/kg of body weight (5). EC has been identified as a carcinogen in species including mice, rats, hamsters and monkeys (6–8), and has been classified as a possible human carcinogen by the International Agency for Research on Cancer (2).

First reported to produce adenomas in the lungs of mice (9), EC was subsequently shown to initiate tumors in a variety of tissues including the skin, liver, mammary gland and lymphoid tissue (10,11). However, lung tumors induced by EC are manifested in 2–6 months, whereas tumors in other tissues required a latent period of about a year (6,12). Although a similar spectrum of tumors is induced by both EC and VC, VC is a more potent carcinogen than EC, producing lung tumors that are 20- to 50-fold greater in number than are induced by EC at similar doses (13,14). Moreover, in comparison with EC, VC induces 3-fold higher levels of DNA adducts in liver and lung (15), has 40 times the clastogenic potency of EC and induces a higher frequency of micronucleated reticulocytes in peripheral blood (16). Recent in vivo studies, using F1 (Big Blue® x A/J) transgenic mice and phage cII as a reporter gene, demonstrated that a dose of EC that was 17-fold greater was required to produce similar mutant frequency (MF) values in the lung as was induced by VC (60 mg/kg, i.p.) (17). These findings are consistent with data showing that VC is a more potent carcinogen than EC, and further that the lung is a highly susceptible target tissue.

Previous studies have postulated that the carcinogenicity of EC is mediated by its metabolism to VC and subsequently to vinyl carbamate epoxide (VCO), a metabolite that has been proposed to be the ultimate carcinogenic species (13,14). Subsequent studies, using human liver microsomes, provided data to support this early proposal and implicated CYP2E1 in the sequential oxidation of EC to VC and ultimately to VCO (18,19). This bioactivation pathway involving CYP2E1 has also been identified in murine and human lung (20–22). Previous studies have used the garlic constituent diallyl sulfone (DASO2) as an efficacious inhibitor of CYP2E1 (23). DASO2 is derived from diallyl sulfide, a component of garlic oil, and it has been estimated that 1 g of garlic yields ~30–100 µg of diallyl sulfide (24). In murine lung, pre-incubation with DASO2 produced a 50% decrease in covalent binding of [14C-carbonyl]VC to microsomal proteins (21). On the basis of these data, we reasoned that inhibition of CYP2E1 by DASO2 will lead also to inhibition of VCO formation, and hence diminish the mutagenic and clastogenic effects evoked by VC (Fig.1). We were interested in expanding our studies with VC and DASO2 in conjunction with a model of mutagenicity developed recently with F1 (Big Blue® x A/J) transgenic mice (17). Here, we have performed in vivo studies to test the hypothesis that CYP2E1-mediated bioactivation of VC plays a central role in the development of mutagenesis and clastogenesis, and further that inhibition of this pathway by DASO2 leads to abrogation or a decrease in their incidences. A primary objective of this investigation was to undertake studies with VC, using F1 (Big Blue® x A/J) transgenic mice and the {lambda} cII-positive selection system (25), to determine the inhibitory effects of DASO2 against the formation of mutations in the lung and small intestine. We have also determined the anti-clastogenic potential of DASO2 in inhibiting micronucleus formation in peripheral blood reticulocytes. Our results demonstrated that pre-treatment with DASO2 significantly reduced VC-induced MFs in both the lung and small intestine, as well as the frequency of micronucleated reticulocytes. These findings demonstrated that bioactivation of VC via CYP2E1 mediates its mutagenic and clastogenic effects, and further that DASO2 inhibits this metabolic pathway and the resultant adverse outcome.


Figure 1
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  		Fig. 1. Proposed scheme of CYP2E1 inactivation by the epoxide of DASO2 and inhibition of mutations from VCO.

 

   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 References
 
Chemicals and reagents
Chemicals were purchased from suppliers as follows: chloroform, NaCl, ethylenediaminetetraacetic acid (EDTA) and phenol:chloroform:isoamyl alcohol (25:24:1) (Fisher Scientific, Fair Lawn, NJ); acridine orange, RNase, sodium dodecyl sulfate, Tris–Base, MgSO4·7H2O, thymine vitamin B1, maltose, kanamycin and JumpStartTM ReadyMixTM REDTaqTM DNA polymerase (Sigma–Aldrich, St Louis, MO); proteinase K and InvitrogenTM PCR purification kit (InvitrogenTM Life Technologies, Carlsbad, CA); BactoTM Agar and BactoTM Tryptone (BD, Sparks, MD) and Stratagene's TranspakTM (Stratagene, La Jolla, CA). DASO2 was synthesized by Colour Your Enzyme (Bath, Ontario, Canada). All other chemicals used were purchased from standard commercial suppliers.

Animals and treatment
All experiments were performed according to a protocol approved by the Animal Care Committee of the Queen's University. Male Big Blue® mice (8–10 weeks of age) were obtained from Stratagene, La Jolla, CA, and female strain A/J mice (6–8 weeks of age) were obtained from Jackson Laboratories, Bar Harbor, ME. The mice were acclimatized to laboratory conditions for 1 week after arrival before breeding, maintained on a 12-h light–dark cycle and provided with food (Mouse Diet 5015; PMI Nutrition International, Brentwood, MO) and water ad libitum. Male Big Blue® mice were bred with female strain A/J mice, and the F1 hybrid offspring were used for all the studies. Eight-week-old F1 (Big Blue® x A/J) mice were randomized into two experimental groups. The first group of mice was used for mutational dose–response studies: male and female mice were treated by gavage with 12.5, 25, 50, 100 or 200 mg/kg of DASO2 in water (0.1 ml). After 2 h, the mice were treated with the maximum tolerated dose of VC (60 mg/kg, i.p.) in saline. The selection of the 2 h period was based on findings from previous time-course studies showing maximal inhibition (75%) of CYP2E1-dependent p-nitrophenol hydroxylation 2 h after DASO2 treatment (26). Tissues were harvested 4 weeks after treatment. Controls for the mutational studies comprised of mice that were (i) treated with the vehicle (untreated), (ii) treated with DASO2 alone or (iii) treated with VC alone. The second group of mice was used for studies of micronucleated reticulocytes: male and female mice were treated with 50, 100 or 200 mg/kg, p.o., of DASO2 in water. After 2 h, the mice were treated with 60 mg/kg, i.p., of VC in saline. Peripheral blood was obtained from the tail vein 48 h after VC treatment and placed on acridine orange-coated slides. The frequencies of micronucleated reticulocytes were determined and scored according to the method of Hayashi et al. (27). The controls consisted of mice that were (i) treated with the vehicle (untreated), (ii) treated with DASO2 alone or (iii) treated with VC alone.

Sampling of tissues
The lungs were excised and rinsed in saline. The entire length of the small intestine was removed, flushed with phosphate-buffered saline and inverted using a thin metal probe. Epithelial suspensions were made from the small intestine by placing the tissue in 3 ml of 75 mM KCl/20 mM EDTA solution and gently forcing it in and out of a 5 cc syringe without a needle. After isolation, all tissues were immediately frozen with liquid nitrogen and then stored at –80°C.

DNA extraction
The tissues (lung and small intestine epithelial suspensions) were incubated overnight in lysis buffer (10 mM Tris, pH 8, 150 mM NaCl and 20 mM EDTA), containing 125 µg/ml of proteinase K, 100 µg/ml RNase and 1% sodium dodecyl sulfate, in a water bath maintained at 55°C. Extraction of DNA was carried out by standard procedures using phenol:chloroform:isoamyl alcohol (25:24:1) and chloroform and precipitated with 100% ethanol. The DNA was collected by spooling with a capillary tube with a hook at one end and stored in 100 µl of Tris–EDTA buffer (10 mM Tris, 1 mM EDTA, pH 8) for 4–5 days at room temperature. The concentration of DNA was determined by measuring the optical density of the samples at 260 nm.

cII mutation assay
The {lambda}LIZ{alpha} shuttle vector containing the cII transgene was recovered by in vitro packaging using Stratagene's TranspakTM packaging reaction system. The hfl– bacterial host strain Escherichia coli G1250 (genotype G1217hflA::Tn5 hflB29 tn10, a spontaneous mutant resistant to a T1-like phage; Stratagene) was grown overnight at 37°C in a shaker in Luria-Bertani broth containing 10 µl/ml of 20% (w/v) maltose:1 M MgSO4 and 1 µl/ml of 25 mg/ml kanamycin stock. The next day, a 10-fold dilution of the overnight culture was made with Luria-Bertani broth and 40 µl/ml of 20% (w/v) maltose:1 M MgSO4, and was incubated at 37°C in a shaker until its OD600 was 0.5. The same day, culture was centrifuged at 4000g for 10 min following which the Luria-Bertani broth was discarded. The bacterial pellet was re-suspended with an equal volume of 10 mM MgSO4.

The first reaction tube containing the packaging extract and 8 µl of dialyzed DNA was incubated for 90 min at 30°C. To the first reaction tube, 12 µl of the second reaction tube was added, and incubated for 90 min at 30°C. The in vitro packaging reaction was terminated using 1000 µl of sodium magnesium buffer. In order to determine the titers, the packaged reaction was diluted 100-fold in sodium magnesium buffer. The titer sample of 50 µl was added to 200 µl of E.coli G1250 and left for 30 min at room temperature for adsorption to take place. Similarly, selection samples were determined by adding 100 µl of the packaged reaction to 200 µl of E.coli G1250 and left for 30 min for adsorption. To both titer and selection samples ~4 ml of Terrific Broth 1 top agar was added and poured into plates made from Terrific Broth 1. The titer plates were incubated at 37°C for 24 h, whereas selection plates were incubated at 24°C for 48 h. The MF was determined by dividing the number of mutant plaques by the total number of plaques, as estimated from the titer plates.

Sequencing
At least one mutant plaque from each of the 10 selection plates from each mouse was sequenced. The mutant plaques formed at 24°C were cored and re-suspended in 400 µl of sodium magnesium buffer. The cII gene was amplified by taking 10 µl of the cored plaque stock as template for polymerase chain reaction (PCR) with the upstream primer sequence: 5'-AATTAAACCACACCTATGGTG-3' and the downstream primer sequence: 5'-CCTCTGCCGAAGTTGAGTATTT-3'. The PCR was conducted with JumpStartTM ReadyMixTM REDTaqTM DNA polymerase in a standard 50 µl total volume. The PCR products (400 bp) were confirmed by standard agarose gel electrophoresis and subsequently purified using Qiagen QIAquick PCR purification kit. The purified PCR products were sequenced by Cortec DNA Services Laboratories (Kingston, Ontario, Canada).

Micronucleus assay
The frequency of micronucleated peripheral blood reticulocytes was determined according to the method of Hayashi et al. (27). Briefly, tail blood was obtained 48 h after VC treatment, which was identified previously as the time of manifestation of maximum frequencies of micronucleated reticulocytes (16). A volume of 5 µl of tail blood was placed on a glass slide pre-coated with 10 µl of 1% acridine orange solution. A cover glass was placed over the blood sample and the blood cells left to stain for 3–15 h. One thousand reticulocytes from each animal were analyzed, using a fluorescence photomicroscope. The method by Hayashi et al. (27) uses acridine orange-coated slides to detect micronuclei in reticulocytes (young RNA-containing erythrocytes). In this assay, a fluorescence photomicroscope equipped with a blue excitation and a 515–530 nm barrier filter was used to distinguish between the micronuclei that fluoresce strongly green and the reticulum structure of reticulocytes that fluoresce red.

Statistical analysis
Data were expressed as mean ± SEM. Data of MFs were analyzed using the Mann–Whitney U-test. The program developed by Cariello et al. (28) was used to analyze differences between the mutation spectra of the various experimental groups. Differences between experimental groups in the micronucleus assay were determined by using one-way analysis of variance and a Bonferroni adjustment. The level of significance for all data analyses was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 References
 
Dose–response studies with DASO2 in lung
All mice survived until the study was completed 4 weeks after the various treatments. Table I shows MF data in the lungs of individual male and female mice (supplementary material is available at Carcinogenesis Online). Since there were no differences in the MFs between male and female mice, the MF values were pooled and treated as a single group of mice. Spontaneous mutations were detected in control mice treated with neither DASO2 nor VC, but the incidences and resultant MFs were low. Treatment of mice with VC alone produced a MF value that was 5-fold of the level in the untreated control (Table I). The MF of mice treated with 12.5 mg/kg of DASO2 and VC was significantly different from mice treated with DASO2 alone, but did not differ significantly from mice treated with VC alone. Increase of the DASO2 dose to 25 mg/kg resulted in a decrease in the MF in VC-treated mice. However, in view of the considerable variability in the data from individual mice in this experimental group, the MFs were not statistically different from mice treated with DASO2 alone as well as from mice treated with VC alone (Table I). Increase of the DASO2 doses to 50 and 100 mg/kg followed by VC generated MFs that were significantly different from mice treated with the same doses of DASO2 and from mice treated with VC alone. Hence, the 50 mg/kg DASO2 dose was regarded as the minimum dose that elicited a significant decrease in MFs. Treatment of mice with 200 mg/kg of DASO2 and VC produced MF data that were similar to mice treated with the same dose of DASO2. These data demonstrated dose-dependent decreases in VC-induced MFs in the lungs of mice pre-treated with DASO2. However, only a MF value similar to that in untreated mice was manifested in mice treated with 200 mg/kg of DASO2 and VC.


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  		Table I. MFs in lungs of male (M) and female (F) control mice and mice treated with DASO2, VC or DASO2 and VC

 
Dose–response studies with DASO2 in small intestine
Details of MF values in the small intestine in individual mice are given in Table II (supplementary material is available at Carcinogenesis Online). As found in the lung, no differences in MF values were observed between male and female mice, and therefore the data were pooled and treated as a single group. As found in the lung, the low MFs observed in control mice indicated that the incidence of spontaneous mutations in the small intestine was minimal. The MF in the small intestine of mice treated with only VC was a significant 3.3-fold higher than the untreated control (Table II). Treatment with 100 or 200 mg/kg of DASO2 and VC elicited MFs that were significantly higher than mice treated with the same doses of only DASO2. However, treatment with 100 mg/kg of DASO2 and VC produced a MF that was not significantly different from mice treated with VC alone. When mice were treated with 200 mg/kg of DASO2 and VC, a significant decrease of 41% was observed, compared with the MF in mice treated with VC alone. Thus, it was only in the case of the 200 mg/kg DASO2 dose that there was a significant reduction in VC-induced MF. Comparisons of the MF data showed no significant differences in the MFs between the lung and small intestine (Tables I and II).


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  		Table II. MFs in small intestine of male (M) and female (F) control mice and mice treated with DASO2, VC or DASO2 and VC

 
Sequencing
Table III presents the distribution of mutations in the cII gene in the lungs of control mice and mice that were treated with DASO2 (200 mg/kg, p.o.), VC (60 mg/kg, i.p.) or DASO2 (200 mg/kg, p.o.) followed by VC (60 mg/kg, i.p.). The most common spontaneous mutation in the lungs of control mice was G:C to A:T transitions, which comprised 58% (21/36) of the total number of mutations present. A high incidence of C to T transitions (9/10, 90%) occurred at CpG sites, which comprised 25% (9/36) of the total number of mutations found in the lungs of the controls. A nearly identical pattern of mutations was observed in the mice treated with DASO2 only where 67% (20/30) of the total number of mutations were G:C to A:T transitions and all the C to T transitions (10/10, 100%) occurred at CpG sites. The major mutations induced in the lung by VC were G:C to A:T (19/73, 26%) and A:T to G:C (21/73, 29%) transitions and A:T to T:A transversions (21/73, 29%). A high percentage of C to T transitions (8/10, 100%) occurred at CpG sites. Treatment of mice with DASO2 together with VC (DASO2/VC) induced G:C to A:T (13/63, 21%) and A:T to G:C (16/63, 25%) transitions and A:T to T:A (19/63, 30%) transversions. The C to T transitions (6/6, 100%) in the DASO2/VC group occurred at CpG sites. Statistical analysis, applying the program developed by Cariello et al. (28), showed that the cII mutation spectra in the lungs of all the experimental groups were not statistically different from one another.


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  		Table III. cII mutations in lungs of control mice and mice treated with DASO2, VC or DASO2 and VC

 
Table IV presents the distribution of mutations in the cII gene in the small intestine of control mice and mice treated with only DASO2, only VC or DASO2 in conjunction with VC (DASO2/VC). In control mice, the most common spontaneous mutation detected was G:C to A:T transitions, which represented 44% (19/43) of the total number of mutations found in the small intestine. A high level of C to T transitions occurred at CpG sites (8/10, 80%), which comprised 19% (8/43) of the total number of mutations analyzed. A nearly identical pattern of mutations was observed in the small intestine of DASO2-treated mice, where 40% (23/57) of the total number of mutations were G:C to A:T transitions and 73% (8/11) of the C to T transitions occurred at CpG sites. In mice treated with VC, the mutations detected were G:C to A:T (19/64, 30%) and A:T to G:C (17/64, 27%) transitions and A:T to T:A (13/64, 20%) transversions. Nearly one-half of the C to T transitions (5/9, 55%) occurred at CpG sites. In mice treated with both DASO2 and VC, the mutations found were G:C to A:T (19/77, 25%) and A:T to G:C (24/77, 31%) transitions and A:T to T:A (16/77, 21%) transversions. Most of the C to T (7/10, 70%) transitions occurred at CpG sites. Statistical analysis revealed that, as found in the lung, the mutation spectra from the various experimental groups were not significantly different from one another. Thus, pre-treatment with DASO2 did not affect the mutation spectra induced by VC in the small intestine.


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  		Table IV. cII mutations in small intestine of control mice and mice treated with DASO2, VC or DASO2 and VC

 
Micronucleus analysis
The frequency of micronucleated reticulocytes is considered as a valid index of in vivo clastogenicity. Details of the frequencies of micronucleated reticulocytes in peripheral blood are given in Table V. There was no significant difference in the frequencies of micronucleated reticulocytes between male and female mice, and therefore the values were pooled and treated as a single group. The frequencies of micronucleated reticulocytes in mice treated with DASO2 at doses of 50–200 mg/kg remained similar to the levels found in untreated control mice (Table V). Compared with the frequency of micronucleated reticulocytes in control mice, treatment with VC (60 mg/kg, i.p.) elicited a significant 6.8-fold increase in the frequency 48 h after treatment. There was no significant difference in the frequencies of micronucleated reticulocytes between untreated mice and mice treated with DASO2 (50 mg/kg, p.o.). The frequencies following treatment with DASO2 and VC were both 4.5-fold higher than those in untreated mice and mice treated with DASO2. However, the frequency in mice treated with DASO2 and VC were a significant 33% less than in VC-treated mice. Increase of the treatment doses of DASO2 to 100 or 200 mg/kg did not augment its inhibitory effects on micronucleus formation; the frequencies of micronucleated reticulocytes remained similar to the level found in mice treated with 50 mg/kg DASO2 (Table V).


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  		Table V. Inhibition of formation of micronucleated reticulocytes in male (M) and female (F) control mice and mice treated with DASO2, VC or DASO2 and VCa

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 References
 
Recent studies have produced evidence to support the involvement of CYP2E1 in VC oxidation (29). Incubation of VC with recombinant CYP2E1 resulted in the generation of VCO, a putative reactive species that reacted with 2'-deoxyadenosine to produce the adduct, 1,N6-ethenodeoxyadenosine ({epsilon}dAS). The {epsilon}dAS adduct was also produced in incubations of VC with murine lung microsomes. The ability of lung microsomes to generate {epsilon}dAS from VC oxidation is of significance because of the propensity of this type of adduct to miscode in DNA transcription (30,31). In mammalian cells, the {epsilon}dAS adduct is associated with base changes such as A -> G and A -> T (32). Significantly, our recent studies have identified high frequencies of A:T -> G:C and A:T -> T:A mutations in the lungs of VC-treated mice (17). Taken together, these findings have shown that CYP2E1-mediated metabolism of VC resulted in formation of VCO that reacted with DNA to produce {epsilon}dAS and mutations. Consistent with the role of CYP2E1 in VC bioactivation, incubation of lung microsomes from DASO2-treated mice with VC in the presence of 2'-deoxyadenosine resulted in a 70% decrease in formation of the {epsilon}dAS adduct (29). In view of the finding that, in addition to CYP2E1, DASO2 also inhibits the CYP2F enzyme (23), it was relevant to determine whether this P450 was implicated in {epsilon}dAS formation. Preliminary studies confirmed that {epsilon}dAS was not detected in incubations of VC with recombinant mouse CYP2F2 or recombinant rat CYP2F4 (29). Hence, the inhibitory effect of DASO2 on {epsilon}dAS production is mediated mainly by CYP2E1, and is ascribed to generation of an epoxide from DASO2, resulting in formation of the heme adduct, N-alkylprotoporphyrin IX, and CYP2E1 inactivation (33,34). In view of the metabolic events induced by VC and the inhibition of {epsilon}dAS formation by DASO2 as well as the link between {epsilon}dAS formation and development of mutations, we postulated that pre-treatment of mice with DASO2 will lead to inhibition of the frequencies of mutations and micronucleated reticulocytes.

Proclivities for development of lung tumors differ in various inbred strains of mice. Strain A/J mice are susceptible, whereas C57BL/6 mice are resistant to lung tumor formation (12,35). This marked difference in susceptibility is associated with enhanced capacities for VC bioactivation in the lungs of the susceptible A/J versus resistant C57BL/6 mice. In comparison with C57BL/6 mice, A/J mice had elevated levels of CYP2E1, exhibited increased covalent binding of VC to lung proteins and generated 70% higher levels of {epsilon}dAS and 3,N4-ethenodeoxycytidine from VC (36). Here, we have crossed Big Blue® mice with a C57BL/6 background with strain A/J mice and used the hybrid offspring in our studies. This hybrid strain has been reported to possess intermediate susceptibility in lung tumor development (37). The {lambda} cII-positive selection system detects mutations in the {lambda} cII transgene, and is embedded in multiple copies in all tissues of Big Blue® mice (25), thus allowing for mutational comparisons between various tissues in the same animal. After DNA extraction, the cII transgene is recovered by packaging the DNA into phage and infecting an hfl– strain (E.coli G1250). Only phage with cII mutations in the {lambda} cII transgene lyse and form plaques at low temperatures (24°C), whereas all infected bacteria lyse and form plaques at 37°C. MF is assessed by calculating the ratio of mutant plaques to the total bacterial cells that were infected, and this ratio is considered a valid index for in vivo mutagenicity. The mutant plaques can be cored and sequenced for the identification of mutations induced by the test compound in the {lambda} cII transgene (25). In this study, the {lambda} cII mutation assay was used to determine the incidences of mutations in the lung and small intestine in F1 (Big Blue® x A/J) mice. Recent time-course studies with VC in the lungs of the F1 (Big Blue® x A/J) have identified 4 weeks as the optimal sampling or manifestation time, which is the minimum amount of time required for the MF to reach a plateau (17). Although the manifestation time in intestinal epithelium has been reported to be one week or less (38), our studies have nonetheless demonstrated dose-dependent formation of mutations from VC at 4 weeks. Hence, VC was found to be as equally mutagenic in the lung as in the small intestine after 4 weeks of treatment at doses of 45, 60 and 75 mg/kg (17) and thus, both tissues were assayed after 4 weeks.

In this study, treatment of mice with VC alone produced a MF in the lung that was 5-fold of the incidence of spontaneous mutations in mice given neither DASO2 nor VC, attesting to the highly mutagenic potential of VC (Table I). Pre-treatment of mice with DASO2 alone did not alter the MF, and hence DASO2 has no mutagenic effect in the lung. However, pre-treatment with DASO2 elicited dose-dependent decreases in VC-induced MF. A 50% decrease in MFs was detected in the lungs of mice pre-treated with DASO2 doses of 25–100 mg/kg, whereas a 70% decrease was detected upon pre-treatment with a dose of 200 mg/kg (Table I). In the small intestine, the MF generated by VC after pre-treatment with 100 mg/kg of DASO2 was similar to the MF induced by treatment with VC alone (Table II). However, when DASO2 pre-treatment was carried out with a dose of 200 mg/kg, the MF was significantly reduced, compared with the MF induced by VC alone. These findings demonstrated that DASO2 exerted an inhibitory effect on the formation of mutations that was more pronounced in the lung than in the small intestine. A DASO2 dose of 25 mg/kg produced a 50% decrease in MF in the lung, whereas a dose of 200 mg/kg produced a 41% decrease in MF in the small intestine. This discrepancy in MF values between the lung and the small intestine may be attributed to the differing levels of CYP2E1 present in the two tissues. In contrast to the lung in which CYP2E1 is a major P450 (39,40), CYP2E1 is present at low levels in the small intestine (41). Immunoblot analysis of the entire small intestine showed low levels of the CYP2E1 protein, which is consistent with a weak CYP2E1 signal from total RNA from small intestinal enterocytes (41). It should be noted, however, that published data regarding the expression of intestinal P450 enzymes are variable, and may be due, in part, to difficulties in microsomal preparation as a result of high levels of proteases in this tissue (42). Despite the low CYP2E1 expression in the small intestine, the MF (8.7 ± 1.3) in this tissue in VC-treated mice was similar to that in the lung (11.4 ± 2.2), suggesting that P450 enzymes other than CYP2E1 might be involved. However, it should be noted that, to the best of our knowledge, the P450-dependent bioactivation of VC and the formation of DNA adducts in the small intestine have not been characterized. Nevertheless, our results have demonstrated that VC is mutagenic, and pre-treatment with DASO2 significantly decreased the MF in the small intestine. These findings supported the contention that, as manifested in the lung, P450 enzymes have also an integral role in VC metabolism in the small intestine.

The mutagenicity of VC in the lung has been attributed to VC oxidation to VCO and the formation of DNA adducts including {epsilon}dAS and 3,N4-ethenodeoxycytidine (15,29,36). The formation of {epsilon}dAS is associated with A:T to G:C transitions and A:T to T:A transversions (30), whereas the formation of 3,N4-ethenodeoxycytidine (31) is associated with G:C to A:T transitions. In agreement with these findings, sequencing of the cII gene revealed three major mutations induced by VC that are similar in the lung and small intestine: (i) G:C to A:T transitions (lung = 26%; small intestine = 30%), (ii) A:T to G:C transitions (lung = 29%; small intestine = 27%) and (iii) A:T to T:A transversions (lung = 29%; = 20%) (Tables III and IV). Similar mutations were also generated in mice treated with VC in conjunction with DASO2: (i) G:C to A:T transitions (lung = 21%; small intestine = 25%) (ii) A:T to G:C transitions (lung = 25%; small intestine = 31%) and (iii) A:T to T:A transversions (lung = 30%; small intestine = 21%). Statistical analysis confirmed that the mutation spectra produced by VC in the lung did not differ from those in the small intestine. Moreover, the mutation spectra found in the lung and small intestine of mice treated with DASO2 alone did not differ from the controls, suggesting that DASO2 did not affect the distribution of the mutations. In addition, the mutation spectra induced by VC did not differ from those generated in the lung and small intestine when the mice were pre-treated with DASO2. These findings demonstrated that DASO2 pre-treatment does not alter the mutation spectra induced by VC in both the lung and small intestine. The effects of VC are associated with not only mutagenic but also clastogenic events. VC has also been shown to induce a high frequency of micronucleated reticulocytes in male B6C3F1 mice (16). In this study, the results demonstrated that VC induced a high frequency of micronucleated reticulocytes, and further that pre-treatment with DASO2 (50 mg/kg) significantly reduced (33%) the incidence of micronucleated reticulocytes (Table V). Treatment with increased doses of DASO2 (100 or 200 mg/kg) elicited no additional inhibitory effects on the incidence of micronucleated reticulocytes produced by VC. These results suggested that the inhibition evoked by the 50 mg/kg dose of DASO2 was the maximum achievable for micronucleus formation.

In summary, our results demonstrated that pre-treatment of mice with DASO2 significantly decreased VC-induced MFs in the lung and small intestine, but did not alter the mutation spectra in either tissue. Pre-treatment with DASO2 also significantly decreased the frequency of micronucleated reticulocytes produced by VC. These findings showed that DASO2 has anti-mutagenic properties in the lung and small intestine, and anti-clastogenic properties in peripheral reticulocytes. Importantly, these results affirmed that VC bioactivation plays a central role in mutagenesis and clastogenesis, and that inactivation of the bioactivation pathway by DASO2 leads to a protective outcome.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
References
 
Supplementary Figures and Tables can be found at http://carcin.oxfordjournals.org/


   Acknowledgments
 
This work was funded by the Canadian Cancer Society through Grant No. 014061 from the National Cancer Institute of Canada. We wish to thank Kathy Collins, Brandie Millen and Gordon Black for their technical assistance.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 

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Received November 11, 2006; revised February 21, 2007; accepted February 27, 2007.


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