Mechanistic Relationships Between DNA Adducts ...

Experimental Lung Research

ISSN: 0190-2148 (Print) 1521-0499 (Online) Journal homepage: http://www.tandfonline.com/loi/ielu20

Mechanistic Relationships Between DNA Adducts, Oncogene Mutations, and Lung Tumorigenesis in Strain A Mice S. Nesnow, J. A. Ross, M. J. Mass & G. D. Stoner To cite this article: S. Nesnow, J. A. Ross, M. J. Mass & G. D. Stoner (1998) Mechanistic Relationships Between DNA Adducts, Oncogene Mutations, and Lung Tumorigenesis in Strain A Mice, Experimental Lung Research, 24:4, 395-405, DOI: 10.3109/01902149809087376 To link to this article: http://dx.doi.org/10.3109/01902149809087376

Published online: 02 Jul 2009.

Submit your article to this journal

Article views: 14

View related articles

Citing articles: 1 View citing articles

Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ielu20 Download by: [University North Carolina - Chapel Hill]

Date: 10 September 2015, At: 09:24

Mechanistic Relationships Between DNA Adducts, Oncogene Mutations, and Lung Tumorigenesis in Strain A Mice

S. Nesnow, J. A. ROSSand M. J. Mass Biochemistry a n d Pathobiology B r a n c h (MD-68), N a t i o n a l H e a l t h a n d Environmental Effects Research Laboratory, U.S. Environmental Protection A g e n c y , Research T r i a n g l e P a r k , N o r t h Carolina, USA

Downloaded by [University North Carolina - Chapel Hill] at 09:24 10 September 2015

G. D. Stoner D i v i s i o n of Environmental H e a l t h Sciences, School of P u b l i c H e a l t h , A r t h u r J a m e s Cancer H o s p i t a l , Ohio State University, Columbus, Ohio, USA

ThiJ paper describes a series o f studies on the lung tumorzgenic activities of polycyclic aromatic tydrocarbom ( P A H J ) in strain A N mice, their ability to f o r m P A H - D N A adducts in lung tissues, and their ability to mutate the Ki-ras oncogene in PAH-induced tumors. Seven P A H s were Jtudied: cyclopenta[cdlpyrene ( C P P ), benzo[alpyrene ( B [ a ] P ) , benzo[b]Jluoranthene ( B [ b ] F ) , dibenz[a,h] anthracene ( D B A ), 5methylchrysene ( 5 M C ) , ben&]aceanthrylene (BCjlA), and dibenzo[a,llpyrene ( D B [ a , l ] P ) . T h e dose--response data f o r the P A H s revealed lootfold dtferences in tumor potency based on dose, with the order of activity D B [ a , l ] P , DBA > B G ] A > 5MC > CPP > B [ a ] P > B [ b ] F . Large dtferences in tumor multiplicity were also observed between &heP A H s . DNA adducts were measured ly 32P-postlabeling techniques on DNA f r o m lungs of mice treated with these PAH’s. D B [ a , l ] P gave syn- and anti-fiord-region dial-epoxide adducts of dAdo and dGuo; DBA gave both bay-region diol-epoxide-dGuo and bisdihydrodiol-epoxide adducts; C P P gave cyclopenta-ringdGuo adductJ; B [ j ] A gave a mixture of cyclopenta-ring-dGuo and -dAdo adducts; 5MC gave a n t i bay-region diol-epoxide-dGuo adducts; B [ a ] P gave bay-region diol-epoxide-dGuo adducts; and B [ b ] F gave 5-tydroxy-B[b]F-diol-epoxide-dGuoadducts. Ki-ras codon 12 and 61 mutation analysiJ of PAH induced tumors was performed using PCR and dideoxy sequencing methodJ. D B [ a , l ] P gave both codon 1 2 and codon 61 mutations. H i g h proportions o f codon 12 T G T mutations f r o m B [ a ] P - , B [ b ] F - and 5MC-, induced tumors and C G T mutations f r o m CPP- and BCjlA-induced tumors were observed. DBA produced no mutations in Ki-ras codons 12 or 61 by direct sequencing. T h e interrelationships between the tumorigenesi.,, DNA adduct, and oncogene mutation data are discussed. Keywords

carcinogenesis, DNA adducts, oncogenes, polycyclic aromatic hydrocarbons

Received 10 February 1998; accepted 10 February 1998. Address correspondence to Stephen Nesnow, PhD, Biochemistry and Pathobiology Branch (MD-68), National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, MD-68 Research Triangle Park, NC 277 11, USA. E-mail : [email protected] We acknowledge the excellent technical support provided by B. Roop, G . Nelson, and A. Galati. Portions of this work were supported by Cooperative Research Agreement CR-816069 from the U.S. EPA with GDS at the Medical College of Ohio, Toledo, OH, and the Ohio State University, Columbus, OH. This manuscript has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. EPA and approved for publication. Mention of trade names or commercial products should not be construed as endorsement or recommendation for use.

Experimental Lung Research, 24: 3 9 5 4 5 , 1998 Copyright 1998 Taylor & Francis 0190-2148/98 $12.00 .OO

+

395

S.Afesnow et el.


396

Polycyclic aromatic hydrocarbons (PAHs) are a pervasive and abundant class of environmental pollutants found in air, water, food, and waste sites. Many have been examined for carcinogenic activity and have demonstrated varying activities depending on their structural characteristics, such as the number of rings, the sites of fusion, extent of condensation, and degree and site of methylation [l, 21. Complex environmental mixtures or exposures containing PAHs, such as aluminium production, coal gasification, tobacco smoke, coke production, iron and steel founding, and soots, have been deemed to be carcinogenic to humans by the International Agency for Research on Cancer (IARC) [3]. Many of the multiringed ( > 3 rings) PAHs have been evaluated by IARC and been deemed as probably carcinogenic to humans [l]. Much of the cancer data for PAHs have come from chronic studies using dermal application to mouse skin with supplemental data from protocols, including mouse skin tumor initiation/promotion, intraperitoneal administration to newborn mice, and intramammary injection in rats. By comparison, there are scant data on lung tumorigenesis induced by this class of carcinogen. I n this study we sought to answer the following questions: What are the lung tumorigenic potencies of these PAHs in strain A/J mouse lung? What are the major routes of metabolic activation of PAHs in strain A/J mouse lung? What are the Ki-ras mutation spectra in lung tumors induced by these PAHs? Can specific mutations in the Ki-ras oncogene be linked to specific PAH-DNA adducts? The strain A/J mouse lung adenoma system was selected because it has been used with success to examine the carcinogenic activity of over 300 chemicals of wide chemical class diversity [4,51. This tumorigenesis system is quite sensitive to PAHs, and lung adenomas can be scored 6-8 months after treatment. In addition, mutations in the Ki-ras dominant transforming protooncogene have been studied from DNA of tumors induced by a wide variety of chemical carcinogens, including several PAHs [6, 71. Seven PAHs benzo[a]pyrene (B[a]P) , benzo[b]fluoranthene (B[b]F) , benzCjlaceanthrylene (BCIA), dibenz[a,h]anthracene (DBA), 5-methylchrysene, (5MC), cyclopenta[cd]pyrene (CPP), and dibenzo[a,l]pyrene (DB[a,l]P) (Figure 1) - were selected for study based on their environmental occurrence and range of carcinogenic activities on mouse skin [8-131.

+

MATERIALS A N D METHODS Chemicals DBA and B[b]F were purchased from Aldrich Chemical (Milwaukee, WI) and B[u]P was purchased from Sigma Chemical (St. Louis, MO) CPP

Mechanistic Relationships in Strain A13 Mice

5-Methylchrysene

397

w Di

benzo[a,l]pyrene


Dibenz[a,hlanthracene

Benz[jlaceanthVlene

Cyclopenta[cd]pyrene

Benzo[b]fluoranthene

Figure 1 Structures of the seven environmental PAHs examined.

and was obtained from A. Gold, University of North Carolina (Chapel Hill, NC) and 5MC was obtained from S. Amin, American Health Foundation (Valhalla, NY). DB[a,l]P and B[j]A were obtained from Midwest Research Institute (Kansas City, KA). DNA adduct standards were obtained from the NCI Chemical Carcinogen Reference Standard Repository. Tricaprylin was purchased from Eastman Kodak (Rochester, NY).

Animal Treatments Male strain A/J mice, 6-8 weeks old, were obtained from Jackson Laboratories (Bar Harbor, ME). Mice were housed in laminar flow rooms in polycarbonate cages and were maintained under standard conditions and received food and water ad libitum. Mice were treated with PAHs dissolved in tricaprylin by single ip administration and 8 months later were killed by cervical dislocation, the lungs were removed, and fixed in 10% neutral buffered formalin, and the surface tumors were counted. A number of tumors from fresh tissues were excised prior to fixation and frozen in liquid nitrogen for subsequent PCR and sequence analyses. Additional groups of mice were treated with PAHs and harvested at time points up to 21 days. Their lungs were removed for subsequent DNA adduct analyses.

398

S. Nesnow et al. Tumor DNA Isolation

Tumor DNA extraction procedures have been reported earlier [141. Briefly, tumor tissue from individual tumors was minced and resuspended in 10 mM Tris-HC1, p H 7.4, containing 400 mM NaCl, 2 mM EDTA, pH 8.0, and 0.6% SDS. The mixture was digested overnight with proteinase K, proteins were precipitated with saturated NaC1, and DNA was precipitated in 100% ethanol.


PCR Amplification of Tumor DNA PCR amplification procedures have been reported earlier [14]. Briefly, a 11 l-bp region of Ki-ras exon 1 was amplified using 20-mer exonic primers, ( K lU, 5’ATGACTGAGTATAAACTTGT-3’; K lL, 5’-CTCTATC GTAGGGTCGTACT-3’). The PCR reactions contained approximately 500 ng genomic template DNA, 5 pmol of each primer, and 200 pM of each of the dNTPs. PCR was performed with 2.5 units of l a g DNA polymerase using 30 cycles of denaturation, annealing, and polymerization.

Analysis of PCR Products and Direct Sequencing Analyses of PCR products and direct DNA sequencing procedures have been reported earlier [141. Briefly, products of amplification were purified by electrophoresis on a 1% agarose gel, the bands corresponding to 11 1 bp were excised and dialyzed, and the DNA was eluted electrophoretically, concentrated, reduced in volume, and lyophilized. The primer (SP4; 5’AAGTGATTCTGAATTAGCTG-3’) was used to sequence the antisense strand of Ki-ras exon 1. The primer was end-labelled using [ Y - ~ ~ P I A Tand P T 4 polynucleotide kinase. The gel-purified template DNA and end-labelled primer were annealed and sequencing reactions were performed using modified T7 DNA polymerase, dithiothreitol, and dNTP/ddNTP termination mixes. Reactions were incubated for 4 minutes at 37°C and then terminated using a formamide-containing dye solution. Samples were analyzed on a gel containing 8% acrylamide and 7 M urea by electrophoresis. The gel containing the samples was dried under vacuum and exposed for at least 18 hours to Kodax XAR-5 film using intensibing screens.

Analysis of DNA Adducts

DNA was extracted from whole lung tissues by the spermine method of Ross et al. [15] and DNA adducts were analyzed from individual lungs by

Mechanistic Relationships in Strain

A13

Mice

399

the 32P-postlabeling assay using the nuclease P I enhancement method [IS]. The solvent systems used for each individual PAH are found in References [14, 16-20]. Separated adducts were visualized by phosphorimagery or by autoradiography. Cochromatography was performed by combining aliquots of the two DNAs of interest (after isolation by the spermine method) and carrying this mixture through the 32P-postlabelingassay.

R ES ULTS


Tumor Induction Groups of 20 male strain A/J mice treated with a single intraperitoneal injection of each PAH produced a dose-related increase in lung adenomas when scored 8 months after treatment (Figure 2). DBA was administered over a dose range of 0-10 mg/kg and DB[a,l]P over a dose range of 0-6 mg/kg. Both gave significant numbers of lung adenomas per mouse at 1.25 mg/kg (DBA) and 1.5 mg/kg (DB[a,l]P) and at higher doses. B[j]A, CPP, and 5MC gave large numbers of adenomas, >90 per mouse, in a dose range of 100-200 mg/kg. B[b]F- and B[a]P-treated animals were dosed up to 200 mg/kg. At 100 mg/kg, B[a]P and B[a]P- and B[b]F-treated animals produced 12.8 and 5.3 lung adenomas per mouse, respectively. Mice treated with tricaprylin (vehicle control) and urethane (positive control, 1000 mg/kg) gave 0.6 and 27.3 lung adenomas per mouse, respectively.

DNA Adduct Identity Identification of individual DNA adducts was accomplished by the 32Ppostlabeling assay, as reported earlier [14, 16-20]. By use of cochromatography with authentic PAH-DNA adduct standards, specific DNA adducts were identified. B[a]P produced three adducts, with 7R,8S,9S-trihydroxy1OR-(N22 ’-deoxyguanosyl)- 7,8,9,10-t etrahydro-B [a]P being the major adduct (Figure 3, B[a]P adduct 2). Two minor B[a]P adducts observed had been previously identified as resulting from the metabolic activation of 9hydroxy-B[a]P (Figure 3 , B[a]P adduct 1) and trans-7,8-dihydroxy-7,8dihydro-B[a]P (Figure 3, B[a]P adduct 3) [21]. B[b]F produced four adducts; the major adduct was identified as a 5-hydroxy-B[b]F-9,10-diol-l1, 12-oxide-2’-deoxyguanosineadduct (Figure 3 , B[b]F adduct 2) [22]. DBA produced five adducts. The major adduct and two minor adducts were from the metabolic activation of trans,trans-3,4,10,1l-tetrahydroxy-3,4,10,11tetrahydro-DBA (DBA-3,4,10,1l-bis-diol) (Figure 3, DBA adducts 3, 4, 5) [23], while two adducts were anti-DBA-3,4-diol-1,2-oxide-N2-[2’-deoxyguanosine] adducts (Figure 3, DBA adducts 1, 2). 5MC produced six 3adducts; only one was identified : 1R,2S,S-trihydroxy-4-(N2-2’-deoxy-

400

S. Nesnow et al.

150 125

-


100

75

-

50

-

25

-

0

I

1

10 Dose, mg/kg

100

Figure 2 Lung adenoma formation in strain AQ mice 8 months after treatment with a single ip administration of B[a]P, B[b]F, DBA, 5MC, CPP, BCflA, and DB[n,l]P. Each point represents mean tumor counts ( k S D bars) for each dose administered to groups of 20 mice. Doses above 10 mg/kg for B[o]P, B[b]F, 5MC, BCjlA, and CPP, above 1.25 mg/kg for DBA, and above 1.5 mg/kg for DB[a,l]P were significantly different than the tricaprylin control at p < 0.05.

guanosyl)-l,2,3,4-tetrahydro-5MC (Figure 3, 5MeC adduct 4).CPP produced four adducts and all of these adducts were CPP-3,4-oxide-2’deoxyguanosine adducts as previously identified (Figure 3, CPP adducts 1-4) [24]. Major Bb]A adducts were identified as a mixture of dGuo and dAdo adducts from the reaction of the cyclopenta-fused ring oxide, B[j’]A- 1,2-0xide (Figure 3, B[j’]A adduct 3). DNA adducts observed in lungs of strain AD mice treated with DB[a,l]P were identified as being derived from the metabolism of DB[a,l]P to its fjord-region diol epoxides through DB[a,l]P-ll, 12-diol [20]. The predominant adduct was identified as an anti-DB[a,l]PDE-dAdo adduct (Figure 3, DB[a,l]P adduct 6 ) . Other major adducts were anti-DB[a, l]PDE-dGuo (Figure 3, DB[a,l]P adduct 3) and syn-DB[a,l]PDE-dGuo adduct (Figure 3, DB[a,ZlP adducts 2) with minor amounts other adducts. No PAH adducts were observed in the DNA from lungs of tricaprylin-treated control mice (Figure 3, control) or mice treated with the noncarcinogen pyrene (Figure 3, pyrene).


Mechanistic Relationships in Strain A13 M i c e

40 1

Figure 3 32P-postlabeling analyses of DNA adducts from strain AQ mice treated with PAHs. Mice were treated with a single ip dose of PAH dissolved in tricaprylin and 3 days later the lungs were removed. The DNA was extracted and subjected to DNA adduct analyses.

DNA Sequence Analysis of Ki-ras Mutations in Strain A/J Lung Tumors Ki-ras codon 12 and 61 mutation analysis of tumors was performed using PCR and dideoxy sequencing methods and has been previously reported [14, 17-20]. I n codon 12, the major mutation type in B[a]P, B[b]F, 5MC, and DB[a,l]P produced tumors was the TGT mutation (Table 1). Significant numbers of tumors exhibited CGT mutations in the CPP, BIjlA, and 5MC treatment groups. All tumors from mice treated with PAHs except DBA

S.Nesnow et al.

402

Table 1 Induction of mutations in the Ki-rus oncogene in lung tumors &om PAH treatment of Strain A 0 mice


Tumors with codon 12 mutation (%) PAH

TGT

B[a]Pa B[b]F" DBA 5MCb CPPP BGlA* DB[a,l]Pb Vehicleb."

56 56 0 50 25 4 28 0

CGT

GAT

GTT

0

19 4 0 0 10 0 0 41

25 36 0 23 15 31 11 23

4 0

27 50 65 6 6

Tumors with codon 61 mutation (%) CAT

CTA

CGA

CAC

0 0

0 0

0

0

0 0

11 6

22 6

17 12

6 6

Note. The wild-type sequence for codon 12 is GGT and for codon 61 is CAA. Blanks indicate no analysis was performed. The percentage of tumors that exhibited a mutation ranged from 50 to 100%. a Percent tumors with mutations based on codon 12 mutations. Percent tumors with mutations based on codon 12 and codon 16 mutations. Data from Reference [19].

exhibited GTT mutations by direct DNA sequencing. Mutations in the DNA from lung tumors from tricaprylin-treated (vehicle control) mice were mainly GAT and GTT. DBA produced no mutations in Ki-rus codons 12 or 61 above spontaneous levels. Codon 61 mutations were sought in the tumors produced by DBA, DB[u,l]P, and 5MC. Only DB[u,l]P produced mutations in codon 12 and codon 61, with CTA and CGA predominant in codon 6 1. Quantitative DNA Adduct Formation Quantitative DNA adduct formation was measured by the 32Ppostlabeling assay. Maximal total DNA adduction occurred 3-10 days after Table 2 Slopes from the plots of PAH dose versus PAH TIDAL values for six PAHs

Standard error

Adjusted

PAH

Slope from the plot: dose versus tidal value (tinol-day/pg DNA)"

BCaP BCblF DBA 5MC CPP DB[a,l]P

113 37.5 219 264 148 1390

3.9 5.0 19.1 51.5 3.69 267

0.994 0.93 1 0.970 0.863 0.998 0.868

RZ

Source. Data taken from References [16, 201. Slope from the fit of the data to the equation:y = 6, bl[x], wherey is the TIDAL value, b, is they intercept, b, is the slope,

+

and x is the dose.


A13

Mice

403


treatment at all doses [16]. Similar DNA adduction, persistence, and decay curves were observed for all PAHs and all doses [16]. The analysis of quantitative DNA adduct formation over time was performed by harvesting groups of mice over a 3-week period and using postlabeling methodology to determine the levels of metabolites. Area under the curve integration was performed on the adduct formation, decay, and persistence curves. This integration was termed, time integrated DNA adduct levels, or TIDAL [lS]. TIDAL levels for six of the seven PAHs were completed (Table 2) [16, 201. The results show that TIDAL levels were linearly related to dose for each PAH with excellent model fits based on the correlation coefficients. Of the group, DB[a,l]P produced the highest TIDAL levels as the slope of the TIDAL versus dose curve was an order of magnitude larger than the five other PAHs.

DISCUSSION The seven PAH studied represent a broad range of different structural types, including angular, condensed, hexacyclic, pentacyclic, and alkylated. Their response as tumorigens in the strain A/J mouse lung systems is also widely varied, suggesting the large dynamic range of response of this mouse to detect carcinogens. Lung tumorigenic PAH activity in strain A/J mice ranges over approximately two orders of magnitude. While B[aJP, B[b]F, DBA, and DB[a,l]P give similar numbers of lung adenomas per mouse at the highest dose tested, the tumor yields for CPP, 5MC, and B[j]A are significantly higher. The metabolic activation of PAHs in strain A/J mouse lung are similar to those responses seen in mouse skin and other rodent tissues [23-28). The major DNA adducts identified include bay region diol epoxide adducts from B[a]P, DBA, and 5MC, a phenolic diol epoxide adduct from B[b]F, cyclopenta-ring adducts from CPP and B[j]A, bisdihydrodiol epoxide adducts from DBA and fjord-region diol epoxide adducts from DB[a,l]P. Guanine was a dominant target for the activated forms of B[a]P, B[b]F, CPP, B[jJA, DBA, 5MC, and DB[a,l]P, while major adenine adducts were detected with DB[a,l]P and B[j]A. The results we have reported for DNA adducts from mouse lung tissues were consistent with the major Ki-ras mutations found in the tumors produced by each PAH. B[a]P, B[b]F, CPP, B[j]A, DBA, 5MC, and DB[a,l]P produced both guanine adducts and mutations at guanine. DB[a,l]P produced both adenine adducts and mutations at adenine. Large numbers of lung adenomas per mouse ( >90) resulted from the treatment of mice with CPP, Bb]A, and 5MC and each of these PAHs induced significant proportions of tumors with C G T mutations at Ki-ras codon 12. The large tumor multiplicities may be explained in several ways: (1) A significant increase in cell proliferation in the lungs of mice may be

S.Nesnow et al.


404

induced by these agents. (2) The gene product from the CGT mutation ( a mutated p21 protein) may be highly growth stimulatory to the tumors selecting for those that have this mutation. The observations could also be a result of a combination of both processes. DBA presents an interesting and unique example of a PAH that can form bay-region diol epoxide-2'-deoxyguanosine adducts but does not induce mutations in Ki-rus codon 12 or 61. DBA also produces significant amounts of DBA-3,4,10,1 l-bis-diol, which can be further activated to forms that bind to DNA. The uniqueness of the bis-diol pathway suggests that mutations within other codons in Ki-rus, or in other lung tumor susceptibility genes that have been recently identified in the strain A/J mouse, may be targets for DBA [29, 301. We reported earlier that when lung adenomas are plotted against TIDAL values for each dose group for the five PAHs, B[a]P, B[b]F, CPP, 5MC, and DB[a,Z]P, the TIDAL versus tumors relationships were similar even when the extent of DNA binding by each PAH was different. This relationship was not consistent for DBA [16, 20). The use of TIDAL analysis reveals an overall similarity between total DNA adduct formation, persistence and repairs, and the ability to include tumors. This is valid for five PAHs of different structural composition that produce different types of DNA adducts and different types of mutations in the Ki-rus oncogene. This suggests that the probability of tumor formation might be related to the extent of total overall DNA damage and its repair rather than specific adducts at specific sites.

REFERENCES 1. IARC. IARC Monographs on the evaluation of carcinogenic risk of chemicals to humans. Polynuclear aromatic compounds, Part 1 : chemical, environmental and experimental data, Vol 32, Lyon, France: International Agency for Research on Cancer; 1983. 2. Harvey RG. Polycyclic Aromatic Hydrocarbons, Chemistry and Carcinogenicity. Cambridge, UK : Cambridge University Press ; 1991. 3. IARC. IARC Monographs on the evaluation of carcinogenic risks to humans. Overall evaluations of carcinogenicity: an updating of the IARC Monographs Volumes 1 to 42, Supplement 7. Lyon, France: International Agency for Research on Cancer; 1987. 4. Shimkin MB, Stoner GD. Lung tumors in mice: application to carcinogenesis bioassay. Adv Cancer Res. 1975;21:1-58. 5. Stoner GD. Lung tumors in strain A mice as a bioassay for carcinogenicity of environmental chemicals. Exp Lung Res. 1991;17:405-423. 6. Reynolds S, Anderson M. Activation of proto-oncogenes in human and mouse lung tumors. Environ Health Perspect. 199;93:145-148. 7. You M, Candrian U, Maronpot RR, Stoner GD, Anderson MW. Activation of the Ki-ras protooncogene in spontaneously occurring and chemically induced lung tumors of the strain A mouse. Proc Natl Acad Sci USA. 1989;86:307&3074. 8. LaVoie EJ, Braley J, Rice JE, Rivenson A. Tumorigenic activity of non-altemant polynuclear aromatic hydrocarbons in newborn mice. Cancer Lett. 1987;34:15-20. 9. Wynder EL, Hoffmann D. A study of tobacco carcinogenesis, VII : the role of higher polycyclic hydrocarbons. Cancer. 1959;12:1079-1086.



A13 Mice

405

10. Habs M, Schmahl D, Misfeld J. Local carcinogenicity of some environmentally relevant polycyclic aromatic hydrocarbons after lifelong topical application to mouse skin. Arch Geschwulstforsch. 1980;50:226-274. 11. Hecht S, Bonidell W, Hoffmann D. Chrysene and methylchrysenes: presence in tobacco smoke and carcinogenicity. J Natl Cancer Inst. 1974;53:1121-1133. 12. Higginbotham S, RamaKrisna N V S , Johansson SL, Rogan EG, Cavalieri EI. Tumor-initiating activity and carcinogenicity of dibenzo[a,llpyrene versus 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene at low doses in mouse skin. Carcinogenesis 1993;14:875-878. 13. Nesnow S, Gold A, Sangaiah R, Slaga TJ. Mouse skin tumor-initiating activity of benzCjlaceanthrylene in SENCAR mice. Cancer Lett. 1993;73:73-76. 14. Mass MJ, Abu-Shakra A, Roop BC, et al. Benzo[b]fluoranthene: tumorigenicity in strain AjJ mouse lungs, DNA adducts, and mutations in the Ki-ras oncogene. Carcinogenesis. 1996; 17:1701-1 704. 15. Ross JA, Nelson GB, Holden KL. DNA isolation from small tissue samples using salt and spermine. Nucleic Acids Res. 1991 ; I 1 :6053. 16. Ross JA, Nelson GB, Wilson KH, et al. Adenomas induced by polycyclic aromatic hydrocarbons in strain AQ mouse lung correlate with time-integrated DNA adduct levels. Cancer Res. 1995;55: 103% 1054. 17. Mass MJ, Jeffers AJ, Ross JR, et al. Ki-ras oncogene mutations in tumors and DNA-adducts formed by benzCjlaceanthrylene and benzo[a]pyrene in the lungs of strain AQ mice. Mol Carcinog. 1993;8:186192. 18. Nesnow S, Ross JA, Nelson G. et al. Cyclopenta[cd]pyrene-induced tumorigenicity, Ki-ras codon 12 mutations and DNA adducts in strain A/J mouse lung. Carcinogenesis. 1994;15:601-606. 19. You L, Wang D, Galati AJ, et al. Tumor multiplicity, DNA adducts and K-ras mutation pattern of 5-methylchrysene in strain AQ mouse lung. Carcinogenesis 1994;15:2613-2618. 20. Prahalad AK, Ross JA, Nelson GB, et al. Dibenzo[a,l]pyrene-induced DNA adduction, tumorigenicity and Ki-ras oncogene mutations in strain A/J mouse lung. Carcinogenesis 1997;18:1955-1963. 21. Ross J, Nelson G, Erexson G, et al. DNA adducts in rat lung, liver, and peripheral blood lymphocytes produced by i.p. administration of benzo[a]pyrene metabolites and derivatives. Carcinogenesis. 1991 ;12 :1953-1955. 22. Weyand EH, Cai ZW, Wu, Y, Rice JE, He ZM, LaVoie EJ. Detection of the major DNA adducts of benzo[b]fluoranthene in mouse skin : role of phenolic dihydrodiols. Chem. Res Toxicol. 1993;6:568577. 23. Fuchs J, Mlcoch J, Platt KP, Oesch F. Characterization of highly polar bis-dihydrodiol epoxide DNA adducts formed after metabolic activation of dibenz[a,h]anthracene formed in vitro. Carcinogenesis. 1993;14:863-867. 24. Beach AC, Agarwal SC, Lambert GR, Nesnow S , Gupta RC. Reaction of cyclopenta[c,d]pyrene-3,4epoxide with DNA and deoxynucleotides. Carcinogenesis. 1993;14:767-771. 25. Gold A, Eisenstadt E. Metabolic activation of cyclopentaCcd1pyrene to 3,4-epoxycyclopental[cd] pyrene by rat liver microsomes. Cancer Res. 1980;40:394&3944. 26. Shali Y, Kwon H, Skipper PL, Tannenbaum SR: Microsomal metabolism of cyclopenta[cd]pyrene: characterization of new metabolites and their mechanism of formation. Chem Res Toxicol. 1992 ;5 :157-1 62. 27. Amin S, Huie K, Balanikas G, Hecht SS, Pataki J, Harvey RG. High stereoselectivity in mouse skin metabolic activation of methylchrysenes to tumorigenic dihydrodiols. Cancer Res. 1987;47:3613-3617. 28. Carmichael PL, Platt KL, She MN, et al. Evidence for the involvement of a bis-diol-epoxide in the metabolic activation of dibenz[a,h]anthracene to DNA-binding species in mice. Cancer Res. 1963;53 :94&948. 29. Chen B, Johanson L, Weist JS, Anderson MW, You M. The second intron of the K-ras gene contains regulatory elements associated with mouse lung tumor susceptibility. Proc Natl Acad Sci USA. 1994;91:158S1594. 30. Herzog CR, Wiseman RW, You M. Deletion mapping of a putative tumor suppressor gene on chromosome 4 in mouse lung tumors. Cancer Res. 1994;54:4007410.