Inhibition of vinyl carbamate-induced lung tumors and Kras2 mutations by the garlic derivative diallyl sulfone

Mutation Research 662 (2009) 16–21

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Inhibition of vinyl carbamate-induced lung tumors and Kras2 mutations by the garlic derivative diallyl sulfone Lya G. Hernandez, Poh-Gek Forkert ∗ Department of Anatomy and Cell Biology, Queen’s University, Kingston, Ontario, Canada K7L 3N6

a r t i c l e

i n f o

Article history: Received 20 August 2008 Received in revised form 29 October 2008 Accepted 24 November 2008 Available online 3 December 2008 Keywords: Diallyl sulfone Lung tumors Kras2 mutations Vinyl carbamate

a b s t r a c t Vinyl carbamate (VC) is derived from ethyl carbamate (EC), a chemical found in alcoholic beverages and fermented foods. The objectives of this study were to characterize the formation of lung tumors induced by VC in F1 (Big Blue® x A/J) mice, and to identify the mutations formed in the Kras2 gene. In addition, we have tested the hypothesis that pretreatment with diallyl sulfone (DASO2 ) inhibits the adverse effects of VC. Mice were treated with VC (60 mg/kg, i.p.) or DASO2 (50 mg/kg, p.o.) 2 h prior to VC (DASO2 /VC). Lung tumor multiplicity was significantly lower (21%) in mice treated with DASO2 /VC than with VC. Lung tumors induced by VC are manifested as solid or papillary tumors, with the latter being regarded as a more malignant phenotype as they demonstrate no growth restrictions. Solid (42%) and papillary tumors (58%) were found in similar proportions in VC-treated mice. The number of papillary tumors was significantly decreased (44.5%) in mice treated with DASO2 /VC, while there was a proportional increase (44.5%) in the number of solid tumors. The number of tumors with mutations in the first and second exon of Kras2 was significantly lower after treatment with DASO2 /VC (7%) than after treatment with VC (61%). The mutations were mainly found in codon 61, and were identified as A → T transversions (31%) and A → G transitions (25%) in the second base, and A → T transversions (12%) in the third base. All of these mutations were significantly reduced by DASO2 pretreatment. The number of tumors containing Kras2 mutations was highest (38%) in the large papillary tumors. Hence, mice treated with DASO2 /VC had decreased frequencies of Kras2 mutations and reduced numbers of small and large papillary tumors, suggesting that activation of the Kras2 gene may be implicated in lung tumor formation and progression. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Vinyl carbamate (VC) is a chemical compound produced from oxidative metabolism of ethyl carbamate (EC) [1,2], a chemical found in alcoholic beverages and fermented food products such as cheese, bread, yogurt and soya sauce [3]. In rodents, EC is a multisite carcinogen that induces tumors in a variety of tissues including the lung [4], skin, liver, mammary gland and lymphoid tissue [5,6]. The lung appears to be a susceptible target for EC and VC carcinogenicity as, in contrast to tumor formation in other tissues that have a latent period of at least a year, lung tumors are manifested in 2–6 months [7,8]. Although the lung tumors induced by EC have been characterized mainly in mice, tumors have also been observed in other species including rats, hamsters [7,9] and non-human primates [10]. The International Agency for Research on Cancer has classified EC as a possible human carcinogen [11]. The carcinogenicity of EC has been attributed to the oxidation of EC to VC and subsequently to the VC epoxide; both oxidative

∗ Corresponding author. Tel.: +1 613 533 2854; fax: +1 613 533 2566. E-mail address: [email protected] (P.-G. Forkert). 0027-5107/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2008.11.013

steps in rat liver are mediated by the cytochrome P450 enzyme, CYP2E1 [12,13]. The VC epoxide, which has been proposed to be the ultimate mutagenic and carcinogenic species, binds to nucleic acids and forms DNA and RNA adducts [1,2,14–16]. More recent findings have also implicated the CYP2E1 enzyme in VC bioactivation in both murine [17] and human [18] lung. Moreover, differences in CYP2E1 levels in various strains of mice have been associated with differing susceptibilities to the adverse effects of VC [19]. Susceptible Strain A/J mice have elevated levels of CYP2E1 and generated 70% higher levels of the DNA adduct 1,N6 -ethenodeoxyadenosine (␧dA) than resistant C57BL/6J mice after VC treatment [19]. More direct evidence for the involvement of CYP2E1 in VC metabolism has been obtained from experiments demonstrating formation of ␧dA in incubations of recombinant CYP2E1 with VC in the presence of 2 -deoxyadenosine [20]. Furthermore, the production of the ␧dA adduct was inhibited by 70% in incubations of VC with lung microsomes from mice pretreated with diallyl sulfone (DASO2 ), a garlic constituent that is an efficacious inhibitor of CYP2E1 [21]. In comparison with mice treated with only VC, treatment of F1 (Big Blue® x A/J) transgenic mice with DASO2 before VC resulted in decreases of 53% and 33% in frequencies of lung mutations and micronucleated reticulocytes in peripheral blood, respectively [22].

L.G. Hernandez, P.-G. Forkert / Mutation Research 662 (2009) 16–21

The inhibitory effects of DASO2 are mediated, in part, via a mechanism whereby CYP2E1 is inactivated by an epoxide formed from CYP2E1-dependent oxidation of DASO2 , leading to production of the heme adduct, N-alkylprotoporphyrin IX [21,23]. Taken together, these findings supported an important role for the CYP2E1 enzyme in VC metabolism. The formation of ␧dA as a result of metabolism of potentially carcinogenic compounds such as vinyl chloride is of significance due to the ability of this type of adduct to miscode in transcription of DNA [24,25]. The propensity for the miscoding in DNA transcription is associated with base changes including A to T transversions and A to G transitions in mammalian cells. In agreement with these findings, our recent in vivo mutagenicity studies, using F1 (Big Blue® x A/J) transgenic mice and phage cII as a reporter gene, have demonstrated elevated levels of A to T transversions and A to G transitions in the lungs of mice treated with VC [22,26]. These data are consistent with the high frequency of VC-induced lung tumors that have an activating A to T transversion in the second base of codon 61 (CAA) of the Kras2 proto-oncogene [27–29]. Given that the ␧dA adduct and associated mutations are formed in the lungs of VC-treated mice [19,20,26,30], that CYP2E1 is a major P450 involved in VC bioactivation [17,18,20], and further that lung tumors in VC-treated mice have an activated Kras2 mutation in codon 61 [27–29], we have undertaken studies herein to further characterize the mutations in the Kras2 gene and the formation of lung tumors as a result of VC treatment. We have also tested the hypothesis that the inhibitory effects of DASO2 results in abrogation or diminution of the carcinogenic and mutagenic effects produced by VC. Our specific objectives were to carry out studies using F1 (Big Blue® x A/J) transgenic mice to: (i) identify the type of mutations in the first and second exon of the Kras2 gene generated by VC, (ii) characterize the lung tumors formed by VC, and (iii) determine the inhibitory effects of DASO2 against VC-induced mutations and lung tumor formation. Our results showed that treatment with both DASO2 and VC resulted in a lower frequency of mutations in exons 1 and 2 of Kras2 in lung tumors. The results further showed that, in comparison to mice treated with only VC, treatment with DASO2 and VC increased survival rates, significantly decreased lung tumor multiplicity and inhibited tumor growth. 2. Materials and methods 2.1. Chemicals and reagents Chemicals were purchased from suppliers as follows: chloroform, NaCl, KCl, EDTA, phenol:chloroform:isoamyl alcohol (25:24:1), glacial acetic acid and Na2 HPO4 (Fisher Scientific, Fair Lawn, NJ); 37% formaldehyde, KH2 PO4 , RNase, sodium dodecyl sulfate, Tris-Base, and JumpStartTM ReadyMixTM REDTaqTM DNA Polymerase (Sigma–Aldrich, St. Louis, MO); InvitrogenTM PCR purification kit (InvitrogenTM Life Technologies, Carlsbad, CA). DASO2 was synthesized by Colour Your Enzyme (Bath, Ontario, Canada). All other chemicals were purchased from standard commercial suppliers. 2.2. Animals and treatment All experiments were performed according to a protocol approved by the Animal Care Committee of Queen’s University. This study is part of a series of studies that have investigated the mutagenicity of VC in Big Blue® transgenic mice [22,26]. These transgenic mice harbor shuttle vectors with reporter genes such as the phage cII, and offer several advantages including the ability for identification and quantitation of mutations in an in vivo mammalian system. We have previously used the cII reporter gene in the Big Blue® mice for identification and characterization of the in vivo mutagenicity of VC [22,26]. For the sake of consistency, the same strain of mice has been used in this study. Male Big Blue® mice of 8–10 weeks of age were obtained from Stratagene (La Jolla, CA), and female Strain A/J mice of 6–8 weeks of age were obtained from Jackson Laboratories (Bar Harbor, ME). The mice were maintained on a 12-h light/dark cycle and provided ad libitum with water and food (Mouse Diet 5015; PMI Nutrition International Inc., Brentwood, MO). The animals were acclimatized to laboratory conditions for 1 week, after which male Big Blue® mice were bred with female A/J mice. Eight-week-old male and female F1 (Big Blue® x A/J) mice were randomized into four treatment groups: (1) control group in which mice were treated with water (p.o.) and 2 h later with saline (i.p.); (2) DASO2 group in which mice were

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treated with 50 mg/kg DASO2 (p.o.) and 2 h later with saline (i.p.); (3) VC group in which mice were treated with water (p.o.) and 2 h later with VC (60 mg/kg, i.p.); and (4) DASO2 /VC group in which mice were treated with 50 mg/kg DASO2 (p.o.) and 2 h later, with a single dose of VC (60 mg/kg, i.p.). The 50 mg/kg dose used in this study was based on previous findings showing that treatment with this DASO2 dose prior to VC reduced the mutant frequency in the lung by 50% ([22]. Increase of the DASO2 dose to 100 mg/kg did not produce greater inhibition of the mutant frequency in VC-treated mice. In addition, treatment with a DASO2 dose of 50 mg/kg significantly decreased clastogenicity, but this inhibitory effect was not augmented by treatment with doses of 100 or 200 mg/kg. The 2 h time-point was selected for the VC treatment as previous studies have found a 75% decrease in p-nitrophenol hydroxylation, a catalytic activity associated with CYP2E1, 2 h after DASO2 treatment [21]. All mice were sacrificed at 25 weeks when the experiments were terminated. 2.3. Tumor collection and histopathology Visible tumors in the lungs were measured and isolated under a dissecting microscope. Tumors were removed carefully to avoid cross-contamination with nontumor tissue. Once the tumor was removed, it was split into two halves; one-half was frozen immediately in liquid nitrogen and stored at −80 ◦ C for DNA analysis and the other half was placed in 70% ethanol for histological analysis. Paraffin sections were prepared and stained with hematoxylin and eosin (H&E) using routine histological procedures. The tumors were classified as solid or papillary tumors according to established criteria [31,32]. The rest of the lung was fixed in Tellyesniczky fluid (61% ethanol, 0.6× phosphate buffered saline, 3.2% formaldehyde, 4% glacial acetic acid). After 48 h, all visible tumors were measured and divided into two groups based on their size and diameter: small (<2 mm) and large (>2 mm). 2.4. Extraction of tumor DNA Lung tumors were incubated overnight in lysis buffer (10 mM Tris pH 8, 150 mM NaCl, 20 mM EDTA), containing 125 ␮g/ml of proteinase K, 100 ␮g/ml RNase, and 1% sodium dodecyl sulfate, in a 55 ◦ C water-bath. 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 left to precipitate in −20 ◦ C for 2 days and collected by removing the ethanol after centrifugation at 4000 × g for 10 min. The DNA sample was resuspended in 30 ␮l of Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 8) and stored for 4–5 days at room temperature. 2.5. Sequencing of the first and second exon of the Kras2 gene Exon 1 from Kras2 was amplified by using 10 ␮l DNA as a template for polymerase chain reaction (PCR) with the upstream primer sequence:  5 -TTTACACACAAAGGTGAGTGTTAAAAT-3 and the downstream primer 5 GCACGCAGACTGTAGAGCAG-3 . Exon 2 from Kras2 was amplified by using 10 ␮l DNA as template for PCR with the upstream primer sequence: 5 -CCAGACTGTGTTTCTCCCTTC-3 and the downstream primer sequence: 5 TCACATGCCAACTTTCTTATTCA-3 . PCR reactions were conducted with JumpStartTM ReadyMixTM REDTaqTM DNA Polymerase in a standard total volume of 50 ␮l. The PCR products were confirmed by standard agarose gel electrophoresis and subsequently purified using Qiagen QIAquick PCR purification kit. The purified products were sequenced by Cortec DNA Services Laboratories Inc. (Kingston, ON). All Kras2 mutations were confirmed by sequencing using forward and reverse primers. 2.6. Statistical analysis Differences in tumor incidence and tumor multiplicity between experimental groups were analyzed using a one-way ANOVA and a Bonferroni adjustment. The Pearson’s Chi square test was used to identify differences in the proportions of lung tumors in the various treatment groups. The program developed by Cariello et al. [33] was used to analyze for statistical differences between the mutational spectra in exons 1 and 2 of Kras2 [33]. Significant differences between experimental groups were set at P < 0.05.

3. Results 3.1. Lung tumor incidence and multiplicity F1 (Big Blue® x A/J) mice in all the experimental groups, with the exception of the VC-treated group, survived for 25 weeks, at which time they were sacrificed. The mortality in mice treated with VC was 31% (19/62). The incidence (number of mice with lung tumors) and multiplicity (mean number of lung tumors per mouse) of lung tumors in the various experimental groups are summarized in Table 1. A tumor incidence of 25% was observed in the control and DASO2 -treated groups, whereas a tumor incidence of 100% was

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Table 1 Lung tumor incidence and multiplicity in control mice and mice treated with DASO2 , VC or DASO2 and VC. Control

DASO2

VC

DASO2 /VC

Dose (mg/kg) Number of mice Number of tumors

0 24 6

50 33 8

60 43 661

50/60 47 575

Tumor rangea TIb TMc

0–1 25 0.25 ± 0.1

0–2 24 0.24 ± 0.9

4–26 100* 15.4 ± 1.1*

4–19 100* 12.2 ± 0.9* , **

488 (74)* 173 (26)*

492 (86)* , ** 83 (14)* , **

Tumor size Small (<2 mm) Large (>2 mm) a b c d * **

Number of tumors 6 (100)d 0 (0)

8 (100) 0 (0)

Number of tumors per mouse in each experimental group. TI: tumor incidence (percentage of mice with lung tumors). TM: tumor multiplicity (mean number of lung tumors per mouse). Values represent mean ± S.E.M. Percentage of total number of lung tumors in the respective groups is given in parentheses. Significantly different (P < 0.01) from control mice and mice treated with DASO2 . Significantly different (P < 0.05) from mice treated with VC.

observed in mice treated with VC or DASO2 and VC. Tumor multiplicities in mice treated with the vehicle (control) (0.25 ± 0.1) or DASO2 (0.24 ± 0.9) were minimal, whereas the multiplicity in the lungs of mice treated with VC was significantly greater (15.4 ± 1.1) than in either of these two groups. The multiplicity in mice treated with DASO2 and VC (12.2 ± 0.9) was also significantly greater than in both control mice and mice treated with DASO2 , but was significantly lower (21%) than in mice treated with VC alone (Table 1). In this study, lung tumors less or greater than 2 mm in diameter were categorized as small or large tumors, respectively. Small tumors ranged in measurement from 0.5–2 mm, whereas large tumors ranged from >2–4.5 mm. All the tumors observed in both control and DASO2 -treated mice were of small size (Table 1). Small and large tumors were found in VC-treated mice, and of a total of 661 lung tumors analyzed, 488 (74%) were small and 173 (26%) were large. On the other hand, treatment with DASO2 and VC altered the tumor distribution: of a total of 575 lung tumors analyzed, 492 (86%) were small and 83 (14%) were large tumors. Hence, compared to the number of lung tumors produced in mice treated with only VC, pretreatment of mice with DASO2 resulted in a small but significant reduction (12%) in the number of large tumors, and a corresponding small increase in the number of small tumors (12%). 3.2. Histopathology and Kras2 mutations A subset of 145 lung tumors from mice treated with VC, 67 small and 78 large, was harvested and each tumor was split into two halves, one-half was sequenced for mutations in exons 1 and 2 of Kras2 and the other half was analyzed histologically (Table 2). All of the lung tumors were benign alveolar or bronchiolar adenomas with either a solid or papillary growth pattern. Solid tumors arise in the alveolar septae and comprise of continuous cords of uniform cuboidal cells with morphological features characteristic of alveolar Type II cells [32]. Papillary tumors arise in the bronchioles and consist of columnar epithelial cells arranged in a tubular or papillary pattern, characteristic of nonciliated Clara cells [34]. In VC-treated mice, the lung tumors exhibited a solid (42%) or papillary (58%) growth pattern (Table 2). When the distribution of the lung tumors was analyzed in terms of size, the small tumors in VC-treated mice were found to be predominantly solid (33.8%), with a lower number that were papillary (12.4%). On the other hand, the large tumors were predominantly papillary (45.5%), and the solid tumors represented a small percentage (8.3%). Hence, small tumors were mainly of the solid type, whereas large tumors were of the papillary type. A subset of 156 lung tumors from mice treated with both DASO2 and VC was also analyzed. The distribution of the

tumors in mice treated with DASO2 and VC differed from that in mice treated with only VC: the tumors were predominantly solid (86.5%) and were less of the papillary type (13.5%). The majority of small lung tumors in this group of mice were solid (45%), while a smaller number (5%) was of the papillary type. Similar to the distribution of the small tumors, the majority of the large tumors were also solid (42%) and a smaller percentage (8%) was of the papillary type. Thus, treatment with DASO2 prior to VC significantly reduced the proportion of both small and large tumors with a papillary growth pattern. In VC-treated mice, mutations of the Kras2 gene were detected in 61% of the tumors analyzed (Table 2). The mutations predominated in both small and large papillary tumors, although the highest proportion (38%) of Kras2 mutations was manifested in the large papillary tumors. The frequency of mutations detected in the small and large solid tumors were similar and were both low (5.5–6.2%). In mice treated with DASO2 and VC, the frequency of Kras2 mutations were markedly inhibited, and were detected only in 7% of the total number of small and large tumors analyzed. The mutations were negligible (0.6%) in the small solid and papillary tumors. The inhibitory effect of DASO2 was also pronounced in the large tumors of both solid (1.3%) and papillary (4.5%) types. Thus, treatment with DASO2 prior to VC significantly reduced the number of lung tumors with Kras2 mutations. Normal lung tissue, which was assayed and used as a control, revealed no mutations in the second exon of Kras2. In mice treated with VC, about 50% (72/145) of the lung tumors developed mutations in the second exon of Kras2, the majority (49%) of which were of the single type. No double mutations were found, while a triple mutation was detected in a large papillary tumor. In mice treated with VC, 61% of the tumors had Kras2 mutations. However, in mice treated with DASO2 and VC, only 7% (11/156) of the tumors sustained mutations. Hence, no double or triple mutations were found in this group of mice. Thus, in comparison with tumors in mice treated with VC, mice treated with DASO2 and VC had a significantly lower number of tumors with mutations in the second exon of Kras2. The lung tumors from mice treated with VC or DASO2 and VC were analyzed for base substitutions in exons 1 and 2. Normal lung tissue, which served as a control, was also analyzed for the presence of base substitutions. No base substitutions were detected in the samples from normal lung tissue, and neither were there any mutations in spontaneous lung tumors from control mice. The distribution of the base substitutions observed in the first and second exon of Kras2 in lung tumors of mice treated with VC or DASO2 and VC are shown in Table 3. Of the 145 lung tumors analyzed in VC-

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Table 2 Association between tumor type, tumor size and Kras2 mutations in lungs of mice treated with VC or DASO2 and VC. Treatment

VC

DASO2 /VC

Lung tumor type

Solid

Papillary

Solid

Papillary

Number of tumorsa Number of small tumors Number of large tumors Small tumors/Kras2 mutations Large tumors/Kras2 mutations

61 (42)b 49 (33.8) 12 (8.3) 8 (5.5) 9 (6.2)

84 (58) 18 (12.4) 66 (45.5) 17 (11.7) 55 (38)

135 (86.5)* 70 (44.9) 65 (41.7)* 1 (0.6)** 2 (1.3)**

21 (13.5)* 8 (5.1)** 13 (8.3)* 1 (0.6)* 7 (4.5)*

Total number of tumors Number of tumors/Kras2 mutations Total number of mice a b * **

145 89 (61) 43

156 11 (7)* 47

Number of lung tumors sequenced for mutations in exons 1 and 2 of Kras2. Percentage of total number of lung tumors in the respective groups is given in parentheses. Significantly different (P < 0.01) from lung tumors in mice treated with VC. Significantly different (P < 0.05) from lung tumors in mice treated with VC.

Table 3 Base substitutions and their respective amino acid changes in exons 1 and 2 of Kras2 in lung tumors of F1 (Big Blue® x A/J) mice treated with VC or DASO2 and VC. Mutation

Codon

Amino acid change

Exon 1 G→A G→T

12, GGT 12, GGT

Gly → Asp Gly → Val

Total base pair substitutions (exon 1) Total number of tumors analyzed Exon 2 C→T CTG → TGC A→G A→T A→T A→C

VCa 10 (7)b 7 (5) 17 (12) 145

50, ACC 50 and 51, ACC TGT 61, CAA 61, CAA 61, CAA 94, CAC

Thr → Ile Thr, Cys → Thr, Ala Gln → Arg Gln → Leu Gln → His His → Pro

1 (0.7) 1 (0.7) 25 (17) 31 (21) 12 (8) 2 (1.4)

DASO2 /VCa 0 (0) 0 (0) 0 (0)* 156

0 (0) 0 (0) 6 (3.8) 4 (2.6) 0 (0) 1 (0.6) 11 (7)*

Total base pair substitutions (exon 2)

72 (49.7)

Distribution of mutations (exons 1 and 2) G:C → A:T A:T → G:C G:C → T:A G:C → C:G A:T → T:A A:T → C:G

12 (8)b 25 (17) 7 (5) 1 (0.7) 43 (30) 3 (2)

0 (0) 6 (4) 0 (0) 0 (0) 4 (2.6) 1 (0.6)

Total mutations (exons 1 and 2)

91 (62.7)

5 (8)*

Total tumors analyzed a b *

145

156

Number of lung tumors with base substitutions. Percentage of lung tumors with base substitutions is given in parentheses. Significantly different (P < 0.01) from mice treated with VC.

treated mice, only a small number of tumors had G → A transitions (7%) and G → T transversions (5%) in codon 12 of the first exon. No mutations in the first exon of Kras2 were detected in tumors from mice treated with DASO2 and VC. In VC-treated mice, the base substitutions in the second exon extended from codons 50–94. In codon 61, 25 tumors (17%) from VC-treated mice had A → G transitions and 43 tumors (29.6%) had A → T transversions. The majority of lung tumors with mutations sustained a deoxyadenosine substitution (Table 3). Also, none of the C → T transitions occurred at CpG sites. All the base substitutions resulted in amino acid changes. In mice treated with DASO2 and VC, the base substitutions in the second exon were found in codons 61 and 94. There were a total of 11 tumors of which 6 had A → G transitions (3.8%) and 4 had an A → T transversion (2.6%) in codon 61, and 1 had A → C transversion in codon 94. Importantly, all the base substitutions in the tumors involved a deoxyadenosine substitution. Moreover, all of the base substitutions in lung tumors of mice treated with DASO2 and VC produced amino acid substitutions. Statistical analysis using the Chi square test revealed a significant decrease in the num-

ber of lung tumors with base substitutions in mice treated with DASO2 and VC, compared to those in mice treated with VC alone (Table 3). The distribution of Kras2 base substitutions in exons 1 and 2 is summarized in Table 3. VC induced primarily A:T → T:A transversions (30%), A:T → G:C transitions (17%), and G:C → A:T transitions (8%). Lung tumors from mice treated with DASO2 and VC had A:T → G:C transitions (4%), A:T → T:A transversions (2.6%), and A:T → C:G transversions (0.6%). With the exception of minor differences, statistical analysis applying the program developed by Cariello et al. [33] revealed no significant differences in the mutational spectra in lung tumors from mice treated with VC or DASO2 and VC. 4. Discussion The carcinogenicity induced by VC is associated with chemical oxidation to the epoxide, a metabolite that has been proposed as the ultimate reactive species, and that is responsible for the formation of the DNA adducts, ␧dA and 3,N4 -ethenodeoxycytidine (␧dC)

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[19,20,30]. The mutations generated by VC were identified in previous studies as base changes associated with the formation of ␧dA (A:T to G:C transitions and A:T to T:A transversions) and ␧dC (G:C to A:T transitions) [22,35]. The mutational events were elucidated in vivo 4 weeks after VC treatment in the lungs of F1 (Big Blue® x A/J) transgenic mice using the bacterial lambda cII transgene [22,26]. Although these data are valid, the relevance of findings from a bacterial gene to a mammalian system is likely to be enhanced were the mutagenicity of an endogeneous gene also examined. Here, we have extended the findings of this previous work in the lambda cII transgene and carried out studies to identify and characterize the mutations generated by VC in the endogeneous Kras2 gene. Kras2 is a proto-oncogene and a lung tumor susceptibility gene that is commonly mutated in 13–64% of VC-induced lung tumors, mainly through an A → T transversion in the second base of codon 61 [27–29,36]. In agreement with these reported findings, our studies have also identified A → T transversions in the second base of codon 61, but in addition we have detected A → G transitions in the second base and A → T transversions in the third base of codon 61 (Table 3). Codon 61 of Kras2 is a GTPase-sensitive site that when mutated results in an activated Ras protein [28,37]. Our results also showed Kras2 activating mutations involving G → A transitions and G → T transversions in codon 12 in lung tumors from VC-treated mice. Other mutations were detected in codons 50 and 51, and codon 94 (Table 3), which are not implicated in an activated Kras2 [37]. The formation of the lung tumors in conjunction with the formation of these mutations (Tables 2 and 3) suggested the involvement of Kras2 activation with the early events and possibly initiation in the development of lung tumors. The mutations in codon 61 of the Kras2 gene produced a substitution of the normal Gln61 with Arg61, Leu61 or His61, and these amino acid changes are believed to reduce the intrinsic GTPase activity of the protein, rendering it resistant to GTPase activity and capable of cellular transformation [38]. Of importance is the Arg61 substitution because of its association with large lung adenocarcinomas and transformed lung cell lines [39]. Consistent with this finding, all of the Arg61 substitutions (Table 3) were found in large papillary tumors, a tumor type associated with malignancy [40]. Furthermore, our findings indicated that deoxyadenosines in the second and third base of codon 61 of Kras2 are susceptible to VCinduced mutations. A deoxyadenosine substitution occurred in 97% (70/72) (Table 3) of the total base substitutions in the second exon of Kras2 in lung tumors of VC-treated mice, thus supporting the contention that the generation of ␧dA by VC metabolism is a critical event in VC-induced lung carcinogenicity. Our recent studies have demonstrated significant inhibition in ␧dA formation in incubations of VC with lung microsomes from DASO2 -treated mice [20]. Furthermore, DASO2 inhibited formation of mutations in the lambda cII transgene in the lungs of F1 (Big Blue® x A/J) mice [22]. In this study, we found a significantly lower number of lung tumors that contained Kras2 mutations with base pair substitutions after DASO2 and VC (7%) than after treatment with only VC (61%) (Table 2). Specifically, DASO2 and VC decreased the number of lung tumors with G → A transitions and G → T transversions in codon 12, and A → G transitions and A → T transversions in codon 61 (Tables 2 and 3), all of which result in Kras2 activation. In addition, the frequency of mutations in the lung tumors was significantly reduced after DASO2 compared to after VC (Table 3). In VC-treated mice, the Kras2 mutations predominated in the papillary tumors (50%), and were considerably lower in the solid tumors (12%) (Table 2). In terms of tumor size, the number of large papillary tumors with Kras2 mutations (38%) in VC-treated mice was higher than in the small papillary tumors (12%). On the other hand, a markedly lower number of large papillary tumors were found to harbor Kras2 mutations (5%) in DASO2 -treated mice. These data suggested an association between tumor size as well as tumor type

with the prevalence of Kras2 mutations. Taken together, these findings suggested that the Kras2 gene is implicated in the formation and growth of the lung tumors induced by VC. The solid and papillary tumors induced by VC have been proposed to be derived from the Type II and Clara cells, respectively [41]. Growth of solid tumors is restricted and regression may occur, whereas papillary tumors continue to grow, represent a more advanced stage of neoplastic transformation, and are more likely to acquire characteristics of carcinomas [40]. In this study, similar numbers of solid and papillary tumors were generated by VC treatment (Table 2). However, DASO2 significantly decreased the formation of small and large papillary tumors, although the decrease in the latter was more pronounced (Table 2). These findings are consistent with data from previous studies showing that CYP2E1 is highly concentrated in the Clara cells [42], that CYP2E1 mediates VC metabolism and the formation of mutations [20,26], and that DASO2 inhibits CYP2E1 and the formation of mutations [22]. These data suggested that CYP2E1 inhibition by DASO2 is an important event leading to the decrease in the formation and progression of the papillary tumors. In contrast, there was a significant increase in the number of solid tumors, and no inhibitory effect was elicited by DASO2 pretreatment (Table 2). This result may be associated with the lack of CYP2E1 expression in the Type II cells [42], suggesting that metabolism of VC in this cell type is mediated by a form of P450 unaffected by DASO2 . In addition, the inhibition of CYP2E1 by DASO2 may produce a compensatory up-regulation of a different P450 involved also in VC metabolism. Alternatively, VC metabolism in the Type II cells may proceed via a different pathway that has not as yet been identified. The decrease in the papillary tumors in conjunction with the increase in the solid tumors may account for a lower difference (21%) than expected in the tumor multiplicity between mice treated with VC vs. VC/DASO2 . Hence, our results suggested that DASO2 inhibited the formation and growth of tumors with a more carcinogenic phenotype, and in this respect, the garlic derivative has a protective effect. In summary, this study has characterized the mutagenic effects of VC in the lungs of F1 (Big Blue® x A/J) mice. Our results demonstrated that VC-induced lung tumors developed Kras2 mutations in codons 12 and 61, both of which were reduced by DASO2 pretreatment. The highest frequency of Kras2 mutations was found in the large papillary tumors in VC-treated mice. Pretreatment with DASO2 produced a marked reduction in the development of large papillary tumors that was concomitant with the decreased frequency of Kras2 mutations, suggesting that activation of the Kras2 gene is implicated in lung tumor progression. Conflicts of Interest The authors declare that there are no conflicts of interest. Acknowledgments We wish to thank Drs. Alexander H. Boag, David P. Lebrun and David J. Hurlbut for assistance with the histopathology of the lung tumors. We also wish to thank John L. Dacosta and Gordon Black for technical assistance. This research has been supported by the Canadian Cancer Society; Grant No. 014061 from the National Cancer Institute of Canada. References [1] G.A. Dahl, E.C. Miller, J.A. Miller, Comparative carcinogenicities and mutagenicities of vinyl carbamate, ethyl carbamate, and ethyl N-hydroxycarbamate, Cancer Res. 40 (1980) 1194–1203. [2] G.A. Dahl, J.A. Miller, E.C. Miller, Vinyl carbamate as a promutagen and a more carcinogenic analog of ethyl carbamate, Cancer Res. 38 (1978) 3793–3804.

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