DNA adduct formation and molecular analysis of in vivo lacI mutations in the mammary tissue of Big Blue® rats treated with 7,12-dimethylbenz[a]anthracene

M.G. Manjanatha1, S.D. Shelton, S.J. Culp, L.R. Blankenship and D.A. Casciano

Department of Health and Human Services, Food and Drug Administration, National Center for Toxicological Research, Divisions of Genetic and Biochemical Toxicology, Jefferson, AR 72079, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Recently we compared the lacI and Hprt mutant frequencies (MFs) and types of mutations in lymphocytes of Big Blue® (BB) rats exposed to 7,12-dimethylbenz[a]anthracene (DMBA) under conditions that result in mammary gland tumors. In this study, we have examined the target mammary tissue for DMBA-induced DNA adducts, lacI MF and types of lacI mutations. Seven-week-old female BB rats were given single doses of 0, 20 or 130 mg/kg DMBA by gavage and the DNA adducts and lacI MFs in the mammary tissue were measured over a period of 14 days and 18 weeks, respectively, following treatment. The lacI MF in the mammary tissue increased for 10 weeks and then remained relatively constant; 130 mg/kg DMBA produced a 14-fold increase in the MF (255 ± 50 x 10–6 p.f.u.) over control MF (18.3 ± 4 x 10–6 p.f.u.). 32P-post-labeling analysis of DNA from mammary tissue and splenic lymphocytes of treated rats revealed two major adducts. Comparison of these adducts with DMBA standards indicated that the adducts formed by DMBA involved both G:C and A:T base pairs. DNA sequencing revealed that the majority of DMBA-induced lacI mutations were base pair substitutions and that A:T->T:A (44% of the independent mutations) and G:C->T:A (24% of the independent mutations) transversions were the predominant types. Furthermore, the mutational results revealed a `hotspot' for a G->T mutation in codon 95 (GTG->TTG) of the lacI gene in mammary tissue. These results suggest that DMBA is highly mutagenic to lacI in mammary tissue and that adducts with both G:C and A:T base pairs participate in forming mutations in DMBA-treated BB rats.

Abbreviations: BB, Big Blue®; DMBA, 7,12-dimethylbenz[a]anthracene; MF, mutant frequency.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
The advent of transgenic models has provided the first opportunity to measure directly spontaneous and chemically induced mutagenesis in different tissues in vivo, including those tissues that may be targets for carcinogenesis. Big Blue® (BB) rats offer potential advantages in genotoxicity and/or carcinogenicity studies because: (i) rats are routinely used in toxicology studies for drug evaluation; (ii) rats succumb to a wide variety of neoplasms, both spontaneous and chemically induced, which are similar in morphology and behavior to those seen in humans (35); (iii) several studies indicate that many tissues of BB rats have lower spontaneous mutant frequencies (MFs) than BB mice (68).

We recently compared the mutations recovered from the lacI transgene with mutations found in the Hprt endogenous gene by determining the MFs and types of mutations in lymphocytes of BB rats following 7,12-dimethylbenz[a] anthracene (DMBA) treatment (1,2). The results showed that although DMBA-induced lacI MFs were 3- to 5-fold higher than Hprt MFs, the Hprt gene was more sensitive than the transgene because the Hprt background MF was an order of magnitude lower than the lacI background MF. The results also demonstrated that the overall mutation profiles were remarkably similar except for the location and orientation of base pair substitution mutations in the DNA strand. In the present study, we examined the target mammary tissue for DNA adducts and the frequency and types of lacI mutations following treatment of BB rats with carcinogenic doses of DMBA. We have chosen DMBA because it is a potent, organ-specific carcinogen that is known to induce mammary gland tumors when female rats are exposed to a single dose (914) and our previous experiments have shown that it is a potent mutagen at both the Hprt (15) and the lacI (1,16) loci.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
DMBA (Sigma Chemical Co., St Louis, MO), DMBA-3,4-dihydrodiol (NCI Chemical Carcinogen Repository, Midwest Research Institute, Kansas City, MO) and [ring-3H]DMBA (1005 mCi/mmol) (Chemsyn Science Laboratories, Lenexa, KS) were purchased from the sources indicated. Chloroform, 8-hydroxyquinoline, Rnace-It, proteinase K, X-gal, BB bottom agar formulation (lot no. 067BBMA), BB top agar formulation, BB RecoveraseTM DNA isolation kit and TranspackTM packaging extract were obtained from Stratagene (La Jolla, CA).

Animals and carcinogen dosing
During the course of the experiments, we followed the recommendations of our Institutional Animal Care and Use Committee for the handling, maintenance, treatment and killing of animals. Seven-week-old female BB rats (Taconic Laboratories, Germantown, NY), homozygous for {lambda}/lacI with 30–40 copies of lacI per rat genome, were treated by single gavage with 0, 20 or 130 mg/kg DMBA in 2 ml of sesame oil. The animals that were treated with 0 and 20 mg/kg DMBA were killed 2, 6, 10, 14 and 18 weeks following treatment, whereas rats that received 130 mg/kg DMBA were analyzed only up to 14 weeks. Following killing, mammary gland tissues from these animals were removed aseptically and processed for the lacI mutation analysis.

DNA extraction from mammary epithelial cells and mammary gland
Six mammary glands and surrounding fat pads were removed from the inguinal region of female rats and stromal epithelial cells were isolated from freshly processed mammary gland tissue using methods described by Allaben et al. (17). Since little or no stromal epithelial cells could be extracted from the frozen mammary glands using collagenase (17), the entire frozen mammary gland was used for DNA extraction. First, the glands were ground into powder in a mortar containing liquid nitrogen. The powdered tissue was further processed to isolate nuclei and the genomic DNA was extracted from the nuclei using the BB RecoveraseTM DNA isolation kit according to the manufacturer's instruction manual. The DNA was then resuspended in TE buffer (10 mM Tris–HCl and 1 mM EDTA, pH 7.5) and quantitated by UV absorption.

lacI mutant assay
DNA extraction, {lambda} packaging and plating for lacI mutant plaques were carried out in a `blocked' manner so as to minimize bias from day-to-day variations in procedure. The lacI-containing {lambda} shuttle vector was recovered by mixing the genomic DNA extracted from both mammary epithelial cells and mammary glands with TranspackTM in vitro {lambda} phage packaging extract as described previously (1). The resulting phages were pre-adsorbed to Escherichia coli SCS-8 cells for 20 min at 37°C, mixed with prewarmed NZY top agar containing 1.5 mg/ml X-gal and poured onto BB medium in 250 mm assay trays. The plates were incubated overnight at 37°C and scored for mutant blue plaques. Color control mutants were included with each plating and the results were accepted only if mutant CM1 could be detected. Packaging and plating were repeated for the DNA samples until at least 5x105 plaques were scored for each data point.

The mutant blue plaques were picked into individual tubes containing 0.5 ml of SM buffer and 50 µl of chloroform. To confirm the mutant phenotype and for future use in DNA sequence analysis, all recovered putative mutant phages from the 250 mm assay plates were diluted 1:100 and replated on 100 mm plates with 3.5 ml of top agarose containing 1.5 mg/ml X-gal. The sectored plaques that were observed were also verified for their phenotype as previously specified (1) and confirmed sectored plaques were scored separately. The lacI MF was calculated by dividing the number of verified mutant plaques by the total number of plaques analyzed.

DNA sequence analysis
pLiz phagemids were rescued from verified mutant {lambda} Liz phages as previously described (16) using the BB Excision kit (Stratagene). Briefly, two bacterial strains XL1-Blue and SOLR were grown overnight, pelleted and resuspended in 10 mM MgSO4 at an OD600 of 1. Excision of {lambda} Liz phages was carried out by mixing mutant {lambda} Liz phage stock with XL1-Blue cells and ExAssist helper phages. After a brief incubation at 37°C, 2x YT medium was added to the mixture and incubated at 37°C for 2–2.5 h while shaking at 400 r.p.m. Following the incubation, the mixture was centrifuged at 4000 g and the recovered phagemids were plated with SOLR cells on LB plates containing 50 µg/ml ampicillin. A single phagemid colony was picked from a LB plate and grown overnight in 3 ml of 2x YT medium at 37°C with shaking. Phagemid DNA was extracted using a plasmid DNA miniprep kit (5 Prime->3 Prime, Boulder, CO). The purified phagemid DNA containing the entire lacI gene (1083 bp) was cycle sequenced using an ABI PRISMTM Dye Terminator kit (Applied Biosystems, Foster City, CA) and the sequencing primers listed in Manjanatha et al. (16). The sequencing reactions were analyzed on an ABI 373 Strech automated DNA sequencer following the procedure outlined by Applied Biosystems. All alterations in DNA sequence were verified at least once.

32P-post-labeling assay
One and 14 days following treatment with 0, 20 or 130 mg/kg DMBA, animals were killed and mammary glands and spleen were removed. Mammary stromal epithelia were isolated using methods described by Allaben et al. (17) and T lymphocytes were isolated from spleen as described by Aidoo et al. (18). DNA was extracted from these tissues by slight modifications of the method reported in Beland et al. (19). DNA adducts were then assayed by 32P-post-labeling using the nuclease P1 enhancement procedure of Reddy and Randerath (20). Approximately 10 µg of DNA were used in each assay. Adducts were separated by thin layer chromatography using the solvents described in Singletary et al. (21). Areas of radioactivity were measured with a Storm 860 Imager (Molecular Dynamics, Sunnyvale, CA).

Total DNA binding was quantified through comparison of the 32P activity associated with the adducts in the sample to the activity produced by standards obtained from the DNA of mammary gland tissue of a rat treated with [ring-3H]DMBA as described previously in Manjanatha et al. (15).

Additional standards were made, as follows, to characterize the structures of 32P-post-labeled adducts. Microsomal incubations were conducted in 1 ml volumes and consisted of 50 mM Tris–HCl (pH 7.3), 3 mM MgCl2, 125 µM NADP+ (Sigma), 4 mM glucose 6-phosphate (Sigma), 0.2 U glucose 6-phosphate dehydrogenase (Sigma), 250 µg of poly(dA-dT), poly(dG-dC) or salmon testes DNA (Sigma), 80 µM DMBA-3,4-dihydrodiol (dissolved in 17 µl of dimethylsulfoxide) and 1 mg of microsomal protein. The microsomes were obtained from the livers of male Sprague–Dawley rats that had been pretreated with 3-methylcholanthrene (22). Following a 2 min preincubation with shaking at 37°C without glucose 6-phosphate dehydrogenase, the enzyme was added and the incubations were continued for 30 min. Aliquots (250 µl) from the incubations were treated with 30 U RNase T1 (Sigma) and 3.5 U RNase A (Sigma) for 30 min at 37°C and then 3.6 U proteinase K (Sigma) for an additional hour. The reactions were then extracted three times with 300 µl of a mixture of chloroform and isoamyl alcohol (24:1). The recovery of poly(dA-dT), poly(dG-dC) and DNA was quantified by UV spectrometry.

Statistical analysis
lacI MF and adduct levels as a function of dose and time after treatment were analyzed by a two-way analysis of variance that included the fixed effects of the time after treatment, the dose and the dosextime interactions. Contrasts were constructed to make the comparisons of interest as well as the effects of each treatment with the control and the P values were adjusted by Holm's modification (23) of the Bonferroni procedure to correct for multiple comparisons. Since the standard deviations of the lacI MFs and adduct levels tended to increase with the magnitude of the response, a logarithmic transformation was performed before conducting the analyses. {chi}2 analysis and the statistical test described by Cariello et al. (24) were used to analyze mutation profiles.


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 Materials and methods
 Results
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lacI mutant frequency in mammary tissue as a function of dose and time after treatment
The DMBA treatments used in this study produced toxicity that killed five treated BB rats: four (20%) from the 130 mg/kg dose group and one (4%) from the lowest dose group (20 mg/kg). Treatments with DMBA resulted in dose- and time-related increases in lacI MF (Tables I and IIGoGo and Figure 1Go). The spontaneous MFs at the lacI locus did not increase with time; the maximum MF of 18.3 ± 4x10–6 was reached ~10 weeks after sesame oil treatment (Table IIGo and Figure 1Go). The MFs for the DMBA-treated rats were 1.3- to 14-fold higher than the control and increased to a maximum (255.6 ± 50x10–6) at 10 weeks following DMBA treatment (Table IIGo and Figure 1Go). However, only the 130 mg/kg dose of DMBA resulted in significant increases in MF over control at all time points (P < 0.05) (Table IIGo). The 20 mg/kg DMBA did not produce a significantly higher lacI MF than the control at any of the time points measured.


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Table I. Raw data for lacI mammary MF in BB rats following exposure to DMBAa for 18 weeks
 

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Table II. Average lacI MF in the mammary tissue of BB rats exposed to DMBA for 18 weeks
 


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Fig. 1. Average lacI MFs measured for 18 weeks in mammary tissue of BB rats treated with 0 ({blacksquare}), 20 (•) or 130 ({blacktriangleup}) mg/kg DMBA. MFs were significantly different from control at P < 0.05 for points marked with an asterisk (*).

 
Sectored plaques were also observed in both the control and DMBA treatment groups (Table IGo), but their frequency was small and it neither increased significantly with dose (P < 0.2) nor differed significantly between treated and control animals (P < 0.3).

lacI mutant sequence analysis
For lacI sequence analysis, we selected 41 mutants from 13 control rats killed 6–18 weeks after treatment with sesame oil (Table IIIGo) and 58 mutants from four rats assayed 10 weeks after treatment with 130 mg/kg DMBA. For each mutant, the 1080 bp lacI gene was sequenced in its entirety. At least one base pair substitution, frameshift mutation or deletion was detected in each mutant. Since some of the mutations were isolated more than once from a single animal, they were assumed to be siblings and considered to represent a single in vivo mutational event. Accordingly, a total of 39 independent control and 41 DMBA-induced mutations were recovered and are listed in Tables IV and VGoGo and summarized in Table VIGo.


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Table III. lacI mutations recovered from control mutant plaques in mammary tissue of BB rats
 

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Table IV. Mutations in the lacI gene of mammary tissues from control BB rats
 

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Table V. Mutations in the lacI gene of mammary tissues from BB rats treated with 130 mg/kg DMBA for 10 weeks
 

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Table VI. Summary of types of lacI independent mutations in the mammary tissue of control and BB rats exposed to 130 mg/kg DMBA
 
Simple base pair substitutions comprised most of the mutations recovered following the control (31/39) and 130 mg/kg DMBA (35/41) treatments (Table IVGo). Most of these mutations (60%) were found within the first 360 bp that contains the negative complementing region of the lacI gene (25). Of the 41 independent DMBA mutations, 35 (85%) were single base pair substitutions and six (15%) were deletions or insertions (Table VIGo). Among single base pair substitutions, 20 mutations (49%) occurred at A:T base pairs while 15 (36%) were at G:C base pairs. The predominant mutation was A:T->T:A transversion (44%, 18/41), followed by G:C->T:A transversion (24%, 10/41). Three G:C->A:T transition mutations were also recovered and only one of these occurred at a CpG site. In addition, there were four single base frameshifts, a 2 bp insertion and a 150 bp deletion, leading to frameshift mutations (15% of the total mutations) (Table VGo, summarized in Table VIGo). Fourteen identical mutations (G:C->T:A at base 95) were recovered from four rats dosed with DMBA (Table VGo). Although this mutation could have come from an in vivo clonal expansion in rats 130A, 130B and 130C, the presence of this mutation in all four treated rats suggests that this site is a potential `hotspot' for DMBA mutation in rat mammary tissue.

The DMBA-induced mutation spectrum was significantly different (P < 0.001) from the lacI control spectrum in mammary tissue (Table IVGo, summarized in Table VIGo). In the spontaneous spectrum, 80% (31/39) of the mutations were simple base pair substitutions. Substitution at G:C accounted for 67% of the mutations while only 13% were at A:T. The most frequent type of mutation was G:C->A:T transition (23/39, 59%), with 70% (16/23) occurring at CpG sites. One of the CpG sites in control animals appeared to have a higher propensity for mutation because a CGA->TGA mutation leading to a stop codon at a CpG site at base 329 was recovered independently in five of the 13 rats used in the study. In addition to base pair substitutions, 20% (8/31) of the total mutations were deletions or insertions: six 1–3 bp insertions and two 1–4 bp deletions (Table IVGo).

DNA adduct analysis
DNA isolated from mammary gland stromal epithelia and spleen lymphocytes were subject to 32P-post-labeling analysis (Figure 2Go). In each tissue, one adduct (adduct a) typically accounted for 47–66% of the radioactivity, a second adduct (adduct b) accounted for 6–22% and additional minor adducts contributed altogether ~10–40%. None of these adducts was found in DNA from control rats (not shown).



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Fig. 2. Storm images of thin layer chromatographic separations of 32P-post-labeled DNA adducts from mammary gland stromal epithelium (A) and spleen lymphocytes (B) of a female BB rat treated with DMBA and from standards made by treating salmon testes DNA (C), poly(dG-dC) (D) and poly (dA-dT) (E) with DMBA-3,4-dihdrodiol in the presence of rat liver microsomes.

 
To determine the identity of the adducts, microsomal incubations were conducted with DMBA-3,4-dihydrodiol in the presence of poly(dA-dT), poly(dG-dC) or DNA. The DNA and oligonucleotides were then purified and analyzed by 32P-post-labeling. DNA from the microsomal incubations gave an intense spot that corresponded to adduct a (Figure 2CGo). In addition, there were at least two minor spots, one of which co-eluted with adduct b. 32P-post-labeling of poly(dG-dC) gave an intense spot (Figure 2DGo) that eluted in the region of adduct a. Analysis of poly(dA-dT) gave one major spot (Figure 2EGo) corresponding to adduct b. Several unidentified minor spots were also observed. 32P-post-labeling of poly(dG-dC) mixed with poly(dA-dT) resulted in two discrete spots migrating in the vicinity of adducts a and b (not shown). These data indicate that adduct a results from reaction with dG (or dC), while adduct b results from adduction of dA (or dT).

Total post-labeled adduct levels were determined in the mammary gland stromal epithelium and spleen lymphocytes (Figure 3A and BGo, respectively) 1 and 14 days after DMBA treatment. There was no significant difference in adduct levels in either the mammary gland stromal epithelium or the splenic lymphocytes with dose or time, however, DNA adduct levels in the spleen lymphocyte tended to decrease 14 days after treatment.




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Fig. 3. (A) Total 32P-post-labeled DNA adducts in mammary gland stromal epithelium from female BB rats treated with 0 (•), 20 ({blacksquare}) or 130 ({blacktriangleup}) mg/kg DMBA. Each point represents the mean ± SD of two rats. (B) Total 32P-post-labeled DNA adducts in spleen lymphocytes from female BB rats treated with 0 (•), 20 ({blacksquare}) or 130 ({blacktriangleup}) mg/kg DMBA. Each point represents the mean ± SD of two rats.

 

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One of the major concerns of using BB rats for mutational analysis is whether or not the inherent properties of the lacI transgene affect the sensitivity of this gene to in vivo mutagenesis. Recently, we addressed this issue by comparing in vivo mutational responses between the lacI and Hprt genes in lymphocytes of BB rats exposed to DMBA (1,2). The results showed that the lacI and Hprt genes of BB rats differ with respect to the kinetics of mutant induction, the magnitudes of both the spontaneous and DMBA-induced MF response and in the ability to detect mutants induced by DMBA exposure. We concluded, however, that although lacI was less sensitive than the Hprt gene for detecting chemical-related increases in MF, it is a reasonable reporter for in vivo mutation (1).

In this study we have extended our investigation to evaluate DMBA-induced DNA adduction and MF and types of lacI mutations in the mammary tissue of BB rats treated with 20 and 130 mg/kg DMBA for up to 18 weeks. The results clearly demonstrate that DMBA produces an increase in MF in the lacI gene for at least 14 weeks following treatment. The 130 mg/kg DMBA-induced lacI MFs demonstrated both time- and dose-dependent increases over the control (P < 0.05) (Table IGo and Figure 1Go).

The increase in lacI MF caused by DMBA treatment was accompanied by DNA adduct formation in mammary epithelial cells (Figure 2AGo). We have also determined DMBA adduct formation in spleen lymphocytes (Figure 2BGo) so that in addition to the lacI mutational responses, the DMBA adduct profiles can be compared between the two tissues. Although mammary epithelial cells are the target tissue for the carcinogenicity of DMBA in the rat and lymphocytes can be considered a surrogate tissue, the initial binding was qualitatively and quantitatively similar in these tissues (Figures 2 and 3GoGo). Singletary et al. (21,26) used 32P-post-labeling to examine the DNA adducts formed in the mammary glands of rats treated with DMBA. Through the use of standards, they characterized adducts from the reaction of dG and dA with the syn and anti isomers of DMBA-3,4-dihydrodiol-1,2-epoxide. Upon conducting microsomal incubations of DMBA-3,4-dihydrodiol with poly(dA-dT) and poly(dG-dC), we also obtained evidence for adduction with dG and dA, with a greater extent of reaction appearing to occur with dG. Caution, however, must be exercised in relating the quantity of 32P incorporated into an adduct spot and the absolute amount of adduct, as the efficiency of labeling can vary greatly between adducts (27). When the total DMBA adduct levels in DNA were compared between splenic lymphocytes and mammary epithelial cells, ~88% of the adducts were removed by 14 days in splenic lymphocytes as compared to only 30% removal in mammary epithelial cells (Figure 3Go). This suggests that DMBA adducts persist longer in the target mammary tissue as compared with lymphocytes.

When lacI responses to DMBA-induced mutagenesis were compared between the target mammary tissue and the surrogate lymphocytes, we found that the average lacI MF in the mammary tissue was slightly lower (20–30%) (Figure 1Go) than the MF in lymphocytes (1). This was surprising, because the mammary epithelial cells are the target tissue for the carcinogenicity of DMBA and, therefore, a higher lacI MF was expected in the mammary tissue than in the surrogate lymphocytes. There may be several reasons for this anomaly and some of them are discussed below.

First of all, tissues may differ in cell proliferation rate and mammary tissue may turn over more slowly than lymphocytes. The higher rate of cell division may increase the likelihood of a shortened time interval for DNA repair leading to more adducts being fixed into mutations. However, in a slowly dividing tissue like the mammary gland, a greater number of DNA adducts can be removed, resulting in fewer mutations (28). Our results support this hypothesis because the DMBA-induced lacI MF in the mammary tissue (Figure 1Go) was slightly lower than the MF in the lymphocytes (1). Furthermore, consistent with the different cell proliferation rates for tissues, our results of a time course of MF induction showed that it took 10 weeks after treatment for DMBA-induced lacI MF to reach its maximum in the slowly dividing mammary tissue (Figure 1Go) as opposed to only 6 weeks following treatment in the more rapidly proliferating lymphocytes (1).

Additionally, the discrepancy in lacI MFs between the target and surrogate tissues may be due to the source of extracted DNA. Since little or no epithelial cells could be extracted from frozen mammary tissues using collagenase (17), DNA was extracted directly from the nuclei of frozen mammary gland tissue. When isolating nuclei from mammary tissue for DNA extraction using Stratagene's Recoverase kit most fatty cells are discarded because they tend to float whereas nuclei form a pellet. However, a small portion of the extracted DNA from the mammary epithelial nuclei may still be contaminated with fatty cell DNA and thus result in an underestimation of mammary epithelial MFs. To address this concern, we extracted DNA from freshly isolated mammary epithelial cells (17) from both control and DMBA-treated rats and determined, for a few samples, the lacI MFs, DMBA adducts and types of lacI mutations. When the results were compared, there were no marked differences in the lacI MFs (Table IGo), the adduct profiles (data not shown) or the types of lacI mutations (data not shown) between DNAs extracted from frozen mammary gland and mammary epithelial cells. However, one striking difference was the poor {lambda} phage rescue efficiency for DNA extracted from the mammary epithelial cells (Table IGo).

Although the spontaneous lacI MF in the mammary tissue of BB rats was lower than previously published spontaneous MFs in lymphocytes (1), liver (8) and Rat2TM cells (16), the types of mutations were remarkably similar. The majority of the control mutants had G:C->A:T transition mutations and 70% of these mutations occurred at CpG dinucleotides (Table VIGo). These transition mutations (C->T) at CpG sites are thought to result from hypermethylation of the lacI transgene by mammalian methylases and subsequent deamination of 5-methylcytosine residues in CpG sites to thymine (2931). The second most frequently observed mutation among our control mutants was A:T->G:C transitions and G:C->T:A transversions, although a few A:T->C:G transversions were also observed. All of these may have originated endogenously by oxidative mechanisms or polymerase errors or other unknown mechanisms (31). Although there was no observed spontaneous mutation `hotspot', a C->T transition mutation at base 329 was recovered in five of the 13 rats used in the study, suggesting a propensity for mutation of this CpG site in mammary tissue (Table IVGo).

The statistical test for mutation spectra described by Cariello et al. (24) indicated that the types of mutations recovered from mammary tissue of DMBA-treated rats were strikingly different from control mutations (P < 0.001; Table VIGo). The most common DMBA-induced mutations were A:T->T:A transversions (44%), followed by G:C->T:A transversions (28%) (Tables V and VIGoGo). The predominance of A:T->T:A and G:C->T:A transversions is consistent with the abundance of DMBA–adenine and DMBA–guanine DNA adducts observed in mammary tissue from DMBA-exposed BB rats (Figure 2Go). However, the distribution of base pair substitutions between A:T (57% of base pair substitutions) and G:C (43% of base pair substitutions) in the DMBA mutational spectrum contrasts with the relative abundance of DMBA–dA (22%) and DMBA–dG (66%) adducts determined in vivo. This observation suggests that DMBA–dA and DMBA–dG adducts produce mutations with markedly different efficiencies in the lacI gene of the mammary tissue or that the 32P-post-labeled quantitation is in error.

DMBA-induced lacI mutations in the mammary tissue did not show any strand bias for A:T (P = 0.1, {chi}2 test) or G:C (P = 0.1, {chi}2 test) mutations (Table VGo). The absence of strand specificity for DMBA-induced mutations is consistent with the presumed lack of expression of the transgene in BB rats and the resultant absence of transcription-coupled repair.

The only other study with DMBA in BB systems besides our previous work in Rat2 cells (16) and in BB rat lymphocytes (1,2) is in the skin of BB mice painted with DMBA (31). The DMBA mutational profiles from these studies were quite similar in many respects: the majority of mutations were base pair substitutions and there were similar frequencies of substitutions at A:T and G:C base pairs. The statistical test for mutation spectra described by Cariello et al. (24), however, indicated that DMBA-induced lacI mutations in the mammary tissue were different from the profile of lacI (P = 0.0012) and Hprt mutations (P = 0.001) in lymphocytes. Despite the species and model differences, however, the DMBA-induced lacI mutation spectrum in the mammary tissue was not different from the lacI spectra in Rat2 cells (P = 0.22) and the skin of BB mice painted with DMBA (P = 0.59). The difference in the lacI spectra between lymphocytes and mammary tissue was mainly attributable to differences in the frequency of G:C->T:A and A:T->G:C mutations recovered from the two tissues. There were more than twice as many G:C->T:A mutations in the mammary gland than in lymphocytes (2). In contrast, A:T->G:C mutations represented 13% of the total mutations in lymphocytes whereas none of this type of mutation was recovered from the mammary tissue (Table VIGo). These results suggest that the types of mutations recovered at the lacI locus are tissue-specific and may be due to differences in the relative mutagenicity of DMBA–dG and DMBA–dA adducts or differences in their repair between tissues.

An additional difference in the lacI mutations induced by DMBA in the mouse skin, rat mammary tissue and Rat2 cells concerns the location of A:T->T:A mutations. DMBA-induced A:T->T:A mutations in BB mouse skin and Rat2 cells occurred disproportionately, with the mutated dA located 3' to a dC. A similar sequence bias for A:T->T:A mutations induced by DMBA was also noted previously for the Hprt gene in the lymphocytes of Sprague–Dawley (32) and BB rats (2). This sequence context bias was not seen in the DMBA-induced lacI mutations in the mammary tissue (P = 0.31, {chi}2 test; only 8/18 A:T->T:A mutations had the mutated dA 3' to a dC; Table VGo) or the lymphocytes of BB rats (2). This bias may be attributable to preferential formation and/or removal of DMBA adducts, however, the absence of this bias for lacI in BB rats but not in BB mice suggests that the location of the transgene in the genome of BB rats may influence these processes.

Another striking difference in the lacI mutations induced by DMBA in the mouse, rat and Rat2 cells concerns the location of potential hotspots for mutation. In this study, out of 41 independent mutations recovered from four treated rats, a dG base at position 95 was mutated 14 times to dT in the four rats used in the study (Table VGo). In mouse skin, rat lymphocytes and Rat2 cells treated with DMBA, there was a high frequency of G->T mutations, but none of these mutations involved base 95 of the lacI gene. These results indicate that there is no common lacI mutational hotspot for DMBA among these samples and the lack of a DMBA-specific hotspot for lacI mutagenesis may be due to inherent differences in the sex or species of animal, differences in tissues or cells examined or in the doses or routes of DMBA exposure.

Finally, several previous studies have shown that DMBA-induced mammary tumors in rats often contain a high frequency of A->T transversion mutations in the second base of codon 61 (CAA) of the H-ras gene (3336). In addition, in DMBA-induced erythroblastic leukemias in Long-Evans rats, the A->T mutation frequency in codon 61 of the N-ras gene was as high as 80% (37,38). This observation suggests that DMBA in rat mammary tumors has a stronger preference for interaction with adenines than guanines, to induce A->T mutations in the ras genes similar to lacI mutations in non-cancer tissues. Based on finding similar mutations between the transgene in non-cancer tissues and endogenous cancer genes in tumors, the lacI gene appears to be a good surrogate to model mutational specificity in biologically important cancer genes.

In summary, DMBA is highly mutagenic to lacI and DMBA–dG and DMBA–dA adducts appear responsible for mutations at A:T and G:C base pairs in mammary tissues. Furthermore, different mutation expression times observed for lymphocytes (1) and mammary tissues suggest that the lacI gene may manifest tissue-specific mutation expression times. Finally, more specific studies are presently underway to evaluate the potential of this system as a surrogate to model mutational specificity in biologically important cancer genes.


    Notes
 
1 To whom correspondence should be addressed Email: mmanjanatha{at}nctr.fda.gov

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    References
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 

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Received July 7, 1999; accepted October 8, 1999.