Carcinogenesis Advance Access originally published online on June 29, 2007
Carcinogenesis 2007 28(11):2391-2397; doi:10.1093/carcin/bgm142
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Comparative mutational profiles of the environmental mammary carcinogen, 6-nitrochrysene and its metabolites in a lacI mammary epithelial cell line
1 Department of Basic Sciences
2 Department of Epidemiology and Health Promotion, College of Dentistry
3 Department of Environmental Medicine, School of Medicine, New York University, New York, NY 10010, USA
4 Department of Pharmacology
5 Department of Biochemistry and Molecular Biology, Penn State College of Medicine, Hershey, PA 17033, USA
* To whom correspondence should be addressed. Tel: +1 717 531 1005; Fax: +1 717 531 7072; Email: kelbayoumy{at}aol.com
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionFundingReferences |
|---|
The dietary and environmental agent, 6-nitrochrysene (6-NC) is a powerful mammary carcinogen and mutagen in rats. It is known to be metabolized by ring-oxidation, nitro-reduction and a combination of the two pathways. In order to determine the ultimate mutagenic metabolites, we have compared the previously determined mutational profile of 6-NC in rat mammary gland [T. Boyiri, et al. (2004) Carcinogenesis, 25, 637–643] with that of five of its known metabolites in the cII gene of lacI mammary epithelial cells in vitro. In vivo, 6-NC gives rise to three major mutations, AT > GC, AT > TA and GC > TA (in decreasing order) which comprise >70% of the mutations. The metabolite whose mutational profile was most similar to that of 6-NC in vivo was trans-1,2-dihydroxy-1,2-dihydro-N-hydroxy-6-aminochrysene (1,2-DHD-6-NHOH-C) which arises from both ring-oxidation and nitro-reduction. However, metabolites arising from either ring-oxidation or nitro-reduction alone exhibited some similarities to mutational profile of 6-NC. These results, taken in conjunction with previous data showing that the major DNA adducts in mammary tissue of rats treated with 6-NC are products of the reaction of 1,2-DHD-6-NHOH-C with guanine and adenine, make a strong case that 1,2-DHD-6-NHOH-C is the ultimate genotoxic metabolite from 6-NC.
Abbreviations: 6-AC, 6-aminochrysene; 1,2-DHD-6-AC, 1,2-dihydroxy-6-aminochrysene; 1,2-DHD-6-NHOH-C, trans-1,2-dihydroxy-1,2-dihydro-N-hydroxy-6-aminochrysene; 1,2-DHD-6-NC, 1,2-dihydro-1,2-dihydroxy-6-nitrochrysene; MEC, mammary epithelial cell; MS, mutational spectrum; N-OH-6-AC, N-hydroxy-6-aminochrysene; 6-NC, 6-nitrochrysene; PCR, polymerase chain reaction
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingReferences |
|---|
Breast cancer is second only to lung cancer as the leading cause of cancer-related deaths in American women (1). In addition to genetic disposition, a significant portion of cancer incidence in USA is related to environmental factors and lifestyles, including diet (reviewed in ref. 2). Environmental pollutants that are known to induce mammary cancer in rodents must be regarded as potential human carcinogens. An example of such environmental carcinogens is the class of nitropolynuclear aromatic hydrocarbons (2); 6-nitrochrysene (6-NC) is a representative example of this class of carcinogens. The remarkable carcinogenic activity of 6-NC as compared with all other nitropolynuclear aromatic hydrocarbons tested so far in the rat mammary gland, its environmental occurrence, the ability of human liver, lung and breast tissues to convert 6-NC into DNA reactive metabolites, as well as the discovery of hemoglobin-6-NC metabolite adducts in humans suggests that 6-NC probably contributes to the development of human breast cancer (3–7). Metabolism and DNA-binding studies in mice and rats have indicated that nitro-reduction and ring-oxidation are involved in the metabolic activation of 6-NC (2). These two pathways are also evident when human tissues are used to catalyze the metabolic activation of 6-NC in vitro (2). The first pathway proceeds via simple nitro-reduction to form N-hydroxy-6-aminochrysene (N-OH-6-AC) that yields three DNA adducts, namely N-(dG-8-yl)-6-AC, N-(dI-8-yl)-6-AC and 5-(dG-N2-yl)-6-AC; the structures of these adducts have been characterized (Figure 1) (8). The second pathway involves a combination of nitro-reduction and ring-oxidation yielding a very reactive electrophile, trans-1,2-dihydroxy-1,2-dihydro-N-hydroxy-6-aminochrysene (1,2-DHD-6-NHOH-C), that leads primarily to the formation of 5-(dG-N2-yl)-1,2-dihydroxy-6-aminochrysene (1,2-DHD-6-AC) and N-(dI-8-yl)-1,2-DHD-6-AC (Figure 1), and smaller fractions of other adducts (9,10). These adducts have been detected in vivo in the rat mammary gland (9,10).
|
Our working hypothesis is that, in the absence of DNA repair, these adducts will cause mutations in important cancer genes (e.g. proto-oncogenes, tumor suppressor genes) and these mutations may lead to the development of breast cancer. Characteristic mutations in human cancer genes can potentially serve as molecular markers of past exposure to specific carcinogens. However, as exposures often arise from complex mixtures, and other factors may also be important in inducing mutations, it is generally difficult to identify ‘signature mutations’ of human carcinogens. Transgenic animals containing retrievable ‘reporter genes’ provide a practical system to identify signature mutations of carcinogens that can be compared with mutational spectra of oncogenes or tumor suppressor genes, and such information may be useful in risk assessment.
In our previous studies, we determined the mutant fraction and mutational spectra in the cII gene in histologically confirmed normal, non-involved and tumor (adenocarcinomas) mammary tissues of female transgenic (Big Blue F344 x Sprague–Dawley) F1 rats treated with 6-NC (9). In order to determine the ultimate mutagen/carcinogen derived from 6-NC, we have synthesized various 6-NC metabolites and compared their mutant fraction and mutational profile in cultured lacI rat mammary epithelial cells (MECs) in vitro. In this study, mutational profile refers to the distribution of the different classes of mutations, and the mutational spectrum (MS) refers the positions of different types of mutations along the cII gene. We hypothesize that the metabolite whose MS is most similar to that of 6-NC in vivo is likely the proximate mutagen. Here, the results are compared with those obtained in vivo in the mammary gland of rats treated with 6-NC (9).
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingReferences |
|---|
Chemicals and reagents
6-NC was synthesized using the procedure described by Newman et al. (11). 6-Aminochrysene (6-AC), 1,2-dihydroxy-6-nitrochrysene, 1,2-DHD-6-AC, N-OH-6-AC and 1,2-DHD-6-NHOH-C were synthesized as reported previously (10,12). RNase A, F12 and Dulbecco's modified Eagle's medium were purchased from Fisher Scientific (Pittsburgh, PA). Proteinase K was purchased from Fisher Scientific. The MEC line from a lacI (BigBlueTM) Fisher 344 rat was kindly provided by David Josephy (University of Guelph, Guelph, Ontario, Canada). The preparation of this line has been described (13).
Treatment conditions
Cells were grown in 10 cm2 petri dishes in F12:Dulbecco's modified Eagle's medium (1:1) + 5% fetal bovine serum. An initial dose–response experiment for single treatments was carried out to determine a dose range with minimal toxicity. In all cases, cell survival was >80% for the concentrations of 6-NC and its metabolites utilized in this study. When treated multiple times, treatments were for 18 h, every other day, except weekends. After three treatments, cells were trypsinized and replated at a density of 2 x 106 cells per dish for subsequent treatment. The lineage of each plate was retained after replating. After four treatments, the cells were grown to 
80% confluence and harvested. The doubling time was 24 h.
Mutagenesis
After treatment of the cells, DNA was extracted using a Recoverase kit (Stratagene, La Jolla, CA) as per the manufacturer's instructions, which involves isolation of nuclei, cell lysis, digestion with proteinase K and RNAse and dialysis on a membrane. Phage packaging was carried out using a phage packaging mix prepared from bacterial strains E. coli NM759 and BHB2688 [generously supplied by Dr Peter Glazer (Yale University School of Medicine, New Haven, CT)] according to the published methods (14).
The cII mutagenesis assay was then employed. The BB® rat from which the MEC cell line was derived contains a lambda shuttle vector that includes the bacterial lacI locus and also the cII gene, which is the target for the mutagenesis studies. This system also obviates the potential for ex vivo mutations that could complicate results. This assay detects mutations at the cII locus and possibly the regulator cI locus (15–19). The cII protein is a positive regulator of gene transcription that controls the decision between lytic or lysogenic development pathways in phage-infected cells. In appropriate Escherichia coli (E. coli 1250) host cells, under specified conditions (25°C) only mutants give rise to phage plaques, whereas at 37°C all infected cells give rise to plaques, providing a phage titer (16–19). The mutant fraction is the ratio of mutant to non-mutant plaques and is the measure of mutagenesis. Each compound was assayed in duplicate plates and each DNA sample was assayed at least twice, for a total of at least four plates per sample.
Amplification and sequencing
Mutants were cored from petri dishes and the agar plug was mixed with 100 µl phage buffer. Ten microliters of the buffer was then spread on a selective plate to confirm mutant phenotype and purify mutant phages. Mutant plaques from 6-NC, 6-NC metabolites and controls were randomly selected from at least two selective plates per compound. The purified mutant plaques were then subjected to amplification and sequencing of the cII gene by polymerase chain reaction (PCR). Sequencing was performed by Roderick Haesevoets, University of Victoria, British Columbia, Canada.
Amplification.
Primer sequences.
Forward: 5'-AAAAAGGGCATCAAATTAAACC-3' and reverse: 5'-CCGAAGTTGAGTATTTTTGCTGT-3'.
Reaction mixture (100 µl reaction).
H2O, 59.1 µl; 100 mM deoxynucleoside triphosphate mix, 1.0 µl; 10x buffer, 10 µl; cII forward primer (10 µM), 2.0 µl; cII reverse primer (10.0 µM), 2.0 µl; 50 mM MgCl2, 3.5 µl; Taq, 2.4 µl; sample, 20 µl and 10x buffer: 100 mM Tris–HCl pH 9.0, 500 mM KCl and 1.0% Triton X-100.
PCR conditions.
94.0°C, 4.0 min; 30 cycles: 95.0°C, 30 s; 55.0°C, 30 s; 72.0°C, 2.5 min and 4.0°C, hold.
Purification.
PCR Product Pre-Sequencing Kit (USB, Cleveland, OH); as per kit instructions.
Sequencing.
Primer sequences.
cII forward: 5'-ACCACACCTATGGTGTATG-3' and cII reverse, 5'-GTCATAATGACTCCTGTTGA-3' (only used to confirm a mutation if the sequence from cII forward primer was not clear).
Reaction mixture.
CEQ Dye Terminator Cycle Sequencing Quick Start Kit (Beckman-Coulter).
PCR conditions.
96.0°C, 5.0 min; 30 cycles: 96.0°c, 20 s; 52.0°C, 25 s; 60.0°C, 4.0 min and 4.0°, hold.
Electrophoresis/base calling/trace generation.
Sequencing was done on a CEQ8000 Capillary Electrophoresis DNA Sequencer (Beckman-Coulter, Fullerton, CA).
Analysis software.
SeqMan II v6.1 (DNASTAR).
Statistics
To compare the mutational profiles of 6-NC in vivo with that of its metabolites, the three major classes of 6-NC in vivo mutations (AT > GC, AT > TA and GC > TA substitutions) plus another group comprised of the minor mutations were compared with the corresponding classes of mutations induced by the 6-NC metabolites in vitro. The MS were compared using Pearson's Chi-square statistic with an exact P value obtained by permutation of the tables. Post hoc comparisons were performed on a pair-wise basis comparing each group to 6-NC using the same statistical methodology. The null hypothesis was that the mutational profile of any compound or condition is not different from that of 6-NC in vivo. Similarities between mutational profiles of the metabolites and 6-NC in vivo were assessed by uncertainty coefficients. The lower the value the more similar is the profile to that of 6-NC in vivo. Calculations were performed using the SPSS version 15 statistical software (Chicago, IL).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingReferences |
|---|
Since the major goal of this study was to determine the ultimate mutagenic metabolite of 6-NC in rat mammary tissue, we attempted to determine which of the several possible 6-NC metabolites had a mutational profile most similar to that of 6-NC in vivo (Table I, Figure 2). Mutations were divided into eight classes consisting of the six possible base pair substitutions, insertions or deletions and more complex mutations, which were designated as ‘other’. Clonal mutations (i.e. identical mutations in plaques obtained from the same plate) were only counted once and are not included in Table I or Figure 2.
|
|
Previously, we reported the mutational profile of 6-NC in mammary tissue of (Big Blue Fisher 344 x CD)F1 rats at a carcinogenic dose of 6-NC (9). This profile and that for the corresponding control are included here for comparison in Table I. The major types of 6-NC-induced mutations in vivo were (in decreasing order) AT > GC, AT > TA and GC > TA substitutions with most of the remaining substitutions at GC base pairs and a small fraction of insertions/deletions. These three types of mutations comprised 72% of the total mutations. The mutational profile of 6-NC in vitro was similar to that of 6-NC in vivo, but some differences were noted. AT > GC and AT > TA mutations alone comprised
75% of the mutations, with almost no mutations at GC base pairs. The difference between the two profiles did not reach significance in a two-tailed Chi-square test (P = 0.15).
The 6-NC metabolite with the mutational profile apparently most similar to that of 6-NC in vivo was 1,2-DHD-6-NHOH-C (a metabolite resulting from nitro-reduction and ring-oxidation). Like 6-NC, it contained 50% AT > GA and AT > AT mutations and slightly smaller fractions of GC > AT and GC > CG mutations. No statistically significant difference in its mutational profile from that of 6-NC was noted (P = 0.50, Chi-square test). 1,2-DHD-6-NC (a metabolite resulting from ring-oxidation of 6-NC) also exhibited a mutational profile similar to that of 6-NC in vivo, with 35 % AT > GA and AT > AT mutations, but with somewhat higher relative percentages of mutations at GC base pairs. Again the difference in profile from that of 6-NC was not significant (P = 0.17). N-OH-6-AC, a metabolite derived from simple nitro-reduction induced almost 70% AT > GA and AT > AT mutations and a smaller fraction of GC > AT mutations. Although the major classes of mutations (particularly the AT > GA transversions) were similar to those induced by 6-NC in vivo, probably because of the very high percentage of mutations at AT base pairs and resulting low percentage GC mutations, the difference in mutational profile from that of 6-NC was nearly significant (P = 0.06). The amino metabolites of 6-NC (6-AC and 1,2-DHD-6-AC) exhibited a major difference from the profiles of 6-NC in vivo and from its nitro and hydroxylamino metabolites; the amino metabolites had much lower fractions of mutations at A:T base pairs (ca. 30%, versus 
50% for the nitro/hydroxylamino compounds). The difference in the mutational profile of AC from that of 6-NC in vivo approached significance (P = 0.09). The mutational profile of the in vivo and in vitro controls exhibited major differences from 6-NC in vivo (P < 0.01) and in vitro (P = 0.01). The in vivo control contained only 10% mutations at AT base pairs and the predominant mutations were GC > AT transitions. The in vitro control also contained a relatively small fraction (30%) of mutations at AT base pairs, and none of the AT > TA transversions characteristic of 6-NC and its metabolites. Here, the major mutations were GC > TA and GC > AT substitutions.
The mutations were for the most part, apparently randomly distributed along the cII gene (Table II). Where multiple identical mutations at the same site were included in the table, they do not represent clonal mutations; rather these resulted from independently treated samples (i.e. separately treated dishes). Several positions were preferentially mutated, but there were no obvious common contextual features flanking these positions.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingReferences |
|---|
This study focused on determining the 6-NC metabolite most likely to represent the ultimate mutagenic (and probably carcinogenic) metabolite of 6-NC. As each mutagen gives rise to a characteristic spectrum of mutations, one approach to this question was to determine the metabolite whose MS was most similar to that of 6-NC in the target tissue (rat mammary). For such an analysis, it is desirable that any further biotransformation of metabolites should be limited, as it complicates interpretation of results. Because extensive metabolism and transport of xenobiotics occurs in whole animals, an in vitro system is more likely to produce a cleaner mutational profile from single 6-NC metabolites than an in vivo system. This and issues of time and costs have led us to investigate the mutational profiles of the metabolites in a cell culture system.
The mutational profiles of 6-NC and its metabolites tended to fall into two classes—those where AT > GC and AT > TA substitutions represented the major contributors to the mutations and those where mutations at GC base pairs made up a majority of the mutations. All the metabolites containing a nitro or hydroxylamino group fell into the first class and the aminochrysenes fell into the second class. Apparently, the presence of the nitro or hydroxylamino group in the chrysene moiety (or chrysene moiety metabolites) is mainly associated with the mutations at AT base pairs, and 6-AC and its dihydrodiol metabolite are mainly associated with mutations at GC base pairs. In previous studies, neither the bay region diol epoxide derived from 6-NC or 6-AC gave the same adducts that had been unequivocally identified in vivo in the rat mammary gland (9). These results indicate that these metabolites are not involved in the overall biological activity of 6-NC. Most likely nitrochrysene and its nitro-containing metabolites are reduced to hydroxylamino metabolites of 6-NC, which are ultimate mutagens, and these give rise to DNA adducts leading to AT > GC and AT > TA mutations and possibly some of the GC > TA mutations (although these could also result from aminochrysenes). Apparently, oxidation of the amino group of 6-AC and 1,2-DHD-6-AC to form hydroxylamines is not an efficient process in MECs, as 6-AC and 1,2-DHD-6-AC were weak mutagens and led to a predominance of mutations at GC base pairs, rather than the high percentages of AT > GC and AT > TA mutations induced by the nitro and hydroxylamino compounds. It seems likely that the 6-AC and 1,2-DHD-6-AC-induced mutations result from other pathways, possibly involving ring-oxidation to form the corresponding diol epoxide.
Although it might be thought that 6-NC would yield the same mutational profile in vivo and in vitro, this was not exactly the case. The mutational profiles were similar but in vitro, nearly all the mutations were AT > GC and AT > TA substitutions, whereas in vivo, a significant fraction of mutations at GC base pairs also occurred. As discussed above, it would be expected that in vivo metabolism would be more complex than in vitro metabolism. Also, the cell culture model uses only one cell type, and this cannot replicate the heterogeneity of the cell types in the mammary tissue in the whole animal (e.g. adipocytes, fibroblasts etc.). In addition to differences in metabolism, different cell types would not be expected to respond equally to DNA damage.
Of all the compounds tested, the one whose mutational profile was most similar to that of 6-NC in vivo was 1,2-DHD-6-NHOH-C. The difference between its mutational profile and that of 6-NC in vivo was very low (P = 0.62) and the uncertainty coefficient for this difference (0.013) was the lowest of any of the compounds tested. Thus, the results of this study are consistent with our working hypothesis that 1,2-DHD-6-NHOH-C is the major ultimate mutagenic metabolite of 6-NC. Our previous observations that 1,2-DHD-6-NHOH-C produces multiple adducts (10) are also consistent with induction of three major types of mutations, since different adducts generally lead to different types of mutations. Although there is considerable overlap in the mutational profile of 6-NC in vivo and 1,2-DHD-6-NHOH-C in vitro, we cannot rule out contributions to the mutational profile of 6-NC by the other metabolites, as these too induce some of the same mutations as 1,2-DHD-6-NHOH-C. One possibly anomalous observation was the lower mutagenic potency of 1,2-DHD-6-NHOH-C than that of 6-NC. This is probably a result of the extreme instability of 1,2-DHD-6-NHOH-C (10). Most of this compound decomposes extracellularly before having a chance to react with cellular DNA. 6-NC, on the other hand, is relatively stable extracellularly, and has appreciable time to permeate the cells and be converted intracellularly to 1,2-DHD-6-NHOH-C.
Taken together, the results of this study appear to be mostly in line with those obtained previously in our laboratory that examined carcinogenicity of 6-NC and some of its metabolites in the rat mammary gland (12). However, in the previous mammary carcinogen bioassay, we did not test 1,2-DHD-6-NHOH-C or N-OH-6-AC. These will be investigated in future studies. These results suggested to us that 1,2-DHD-6-NC is the proximate carcinogen and based on the levels and structures of various 6-NC-DNA adducts found in the mammary gland of rats treated with 6-NC, we hypothesized that 1,2-DHD-6-NHOH-C is the ultimate carcinogenic metabolite. These studies, which were carried out using both tritium and 32P-post-labeling, revealed five adducts in mammary tissue from rats treated with 6-NC, with the major identified adduct resulting from 1,2-DHD-6-NHOH-C (10). However, both guanine and inosine adducts were identified, in addition to an unidentified adduct (9,10). Presumably, the inosine adduct resulted from deamination of an adenine adduct. Twenty-four hours after a single oral dose of 50 µmol per rat, adduct levels of 10–6 to 10–7 per nucleotide were observed as judged by 32P-post-labeling levels (9).
As described above, the results demonstrate that the mutation spectrum of this metabolite in vitro is similar to that observed for 6-NC in vivo, further supporting our hypothesis that 1,2-DHD-6-NHOH-C is the ultimate mutagen/carcinogen. Also, the observation of mutations at both GC and AT base pairs is in accord with the DNA adducts derived from 6-NC. Both 6-NC-deoxyguanosine and deoxyinosine (presumably derived from deoxyadenosine) adducts were detected both in in vitro and in vivo systems (7–10). Although it is not possible from this study to definitively assign specific mutations to particular adducts, we hypothesize that the major guanine adduct, 5-(dG-N2-yl)-1,2-DHD-6-AC leads to the GC > TA transversions, based on the similarity of its structure to that of the major benzo(a)pyrene adduct which is reported to induce these transitions (20). The AT > GC and AT > TA mutations presumably arise from the deoxyinosine adducts; we have no information as to the mutational specificities of these individual adducts. The in vitro system employed in the present study represents a relatively rapid assay and can provide important information on the role of metabolites in the mutagenicity and/or carcinogenicity of environmental pollutants.
|
Funding |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingReferences |
|---|
National Cancer Institute (CA 35519).
|
Acknowledgments |
|---|
Conflict of Interest Statement: None declared.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingReferences |
|---|
- Jemal A, et al. Cancer statistics, 2006. CA Cancer J. Clin. (2006) 56:106–130.
[Abstract/Free Full Text] - Amin S, et al. Tumorigenicity of polycyclic aromatic hydrocarbons. On the possible contribution of PAHs and their nitro-derivatives to the development of human breast cancer. In: The Carcinogenic Effects of Polycyclic Aromatic Hydrocarbons, Chapter 9—Luch A, ed. (2005) Imperial College Press. 315–351.
- IARC. Diesel and gasoline engine exhausts and some nitroarenes. IARC Monogr. Eval. Carcinog. Risks Hum. (1989) 46:1–458.[Medline]
- Zwirner-Baier I, et al. Polycyclic nitroarenes (nitro-PAHs) as biomarkers of exposure to diesel exhaust. Mutat. Res. (1999) 441:135–144.[Web of Science][Medline]
- El-Bayoumy K, et al. Induction of mammary cancer by 6-nitrochrysene in female CD rats. Cancer Res. (1993) 53:3719–3722.
[Abstract/Free Full Text] - Chae YH, et al. Roles of human hepatic and pulmonary cytochrome P450 enzymes in the metabolism of the environmental carcinogen 6-nitrochrysene. Cancer Res. (1993) 53:2028–2034.
[Abstract/Free Full Text] - Boyiri T, et al. Metabolism and DNA binding of the environmental pollutant 6-nitrochrysene in primary culture of human breast cells and in cultured MCF-10A, MCF-7 and MDA-MB-435s cell lines. Int. J. Cancer (2002) 100:395–400.[CrossRef][Web of Science][Medline]
- Delclos KB, et al. Identification of C8-modified deoxyinosine and N2- and C8-modified deoxyguanosine as major products of the in vitro reaction of N-hydroxy-6-aminochrysene with DNA and the formation of these adducts in isolated rat hepatocytes treated with 6-nitrochrysene and 6-aminochrysene. Carcinogenesis (1987) 8:1703–1709.
[Abstract/Free Full Text] - Boyiri T, et al. Mammary carcinogenesis and molecular analysis of in vivo cII gene mutations in the mammary tissue of female transgenic rats treated with the environmental pollutant 6-nitrochrysene. Carcinogenesis (2004) 25:637–643.
[Abstract/Free Full Text] - El-Bayoumy K, et al. Identification of 5-(deoxyguanosin-N2-yl)-1,2-dihydroxy-1,2-dihydro-6-aminochrysene as the major DNA lesion in the mammary gland of rats treated with the environmental pollutant 6-nitrochrysene. Chem. Res. Toxicol. (2004) 17:1591–1599.[CrossRef][Web of Science][Medline]
- Neumann MS, et al. The orientation of chrysene. J. Org. Chem. (1940) 5:618–622.[CrossRef][Web of Science]
- El-Bayoumy K, et al. Comparative tumorigenicity of the environmental pollutant 6-nitrochrysene and its metabolites in the rat mammary gland. Chem. Res. Toxicol. (2002) 15:972–978.[CrossRef][Web of Science][Medline]
- McDiarmid HM, et al. Epithelial and fibroblast cell lines cultured from the transgenic BigBlue rat: an in vitro mutagenesis assay. Mutat. Res. (2001) 497:39–47.[Web of Science][Medline]
- Hohn B. In vitro packaging of lambda and cosmid DNA. Methods Enzymol. (1979) 68:299–324.[Medline]
- Jakubczak JL, et al. Analysis of genetic instability during mammary tumor progression using a novel selection-based assay for in vivo mutations in a bacteriophage transgene target. Proc. Natl Acad. Sci. USA (1996) 93:9073–9078.
[Abstract/Free Full Text] - Gollapudi BB, et al. Hepatic lacI and cII mutation in transgenic (lambdaLIZ) rats treated with dimethylnitrosamine. Mutat. Res. (1998) 419:131–135.[Web of Science][Medline]
- Zimmer DM, et al. Comparison of mutant frequencies at the transgenic lambda LacI and cII/cI loci in control and ENU-treated Big Blue mice. Environ. Mol. Mutagen. (1999) 33:249–256.[CrossRef][Web of Science][Medline]
- Watson DE, et al. Spontaneous and ENU-induced mutation spectra at the cII locus in Big Blue Rat2 embryonic fibroblasts. Mutagenesis (1998) 13:487–497.
[Abstract/Free Full Text] - Swiger RR. Just how does the cII selection system work in Muta Mouse? Environ. Mol. Mutagen. (2001) 37:290–296.[CrossRef][Web of Science][Medline]
- Mackay W, et al. Mutagenesis by (+)-anti-B[a]P-N2-Gua, the major adduct of activated benzo[a]pyrene, when studied in an Escherichia coli plasmid using site-directed methods. Carcinogenesis (1992) 13:1415–1425.
[Abstract/Free Full Text]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||