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
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Abstract |
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Abbreviations: BB, Big Blue®; DMBA, 7,12-dimethylbenz[a]anthracene; MF, mutant frequency.
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Introduction |
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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.
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Materials and methods |
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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 /lacI with 3040 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 TrisHCl and 1 mM EDTA, pH 7.5) and quantitated by UV absorption.
lacI mutant assay
DNA extraction, 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
shuttle vector was recovered by mixing the genomic DNA extracted from both mammary epithelial cells and mammary glands with TranspackTM in vitro
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 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
Liz phages was carried out by mixing mutant
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 22.5 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 TrisHCl (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 SpragueDawley 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. 2 analysis and the statistical test described by Cariello et al. (24) were used to analyze mutation profiles.
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Results |
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lacI mutant sequence analysis
For lacI sequence analysis, we selected 41 mutants from 13 control rats killed 618 weeks after treatment with sesame oil (Table III) 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 V
and summarized in Table VI
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The DMBA-induced mutation spectrum was significantly different (P < 0.001) from the lacI control spectrum in mammary tissue (Table IV, summarized in Table VI
). 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 13 bp insertions and two 14 bp deletions (Table IV
).
DNA adduct analysis
DNA isolated from mammary gland stromal epithelia and spleen lymphocytes were subject to 32P-post-labeling analysis (Figure 2). In each tissue, one adduct (adduct a) typically accounted for 4766% of the radioactivity, a second adduct (adduct b) accounted for 622% and additional minor adducts contributed altogether ~1040%. None of these adducts was found in DNA from control rats (not shown).
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Total post-labeled adduct levels were determined in the mammary gland stromal epithelium and spleen lymphocytes (Figure 3A and B, 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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Discussion |
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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 I and Figure 1
).
The increase in lacI MF caused by DMBA treatment was accompanied by DNA adduct formation in mammary epithelial cells (Figure 2A). We have also determined DMBA adduct formation in spleen lymphocytes (Figure 2B
) 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 3
). 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 3
). 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 (2030%) (Figure 1) 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 1) 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 1
) 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 I), 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
phage rescue efficiency for DNA extracted from the mammary epithelial cells (Table I
).
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:CA:T transition mutations and 70% of these mutations occurred at CpG dinucleotides (Table VI
). 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 IV
).
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 VI). The most common DMBA-induced mutations were A:T
T:A transversions (44%), followed by G:C
T:A transversions (28%) (Tables V and VI
). The predominance of A:T
T:A and G:C
T:A transversions is consistent with the abundance of DMBAadenine and DMBAguanine DNA adducts observed in mammary tissue from DMBA-exposed BB rats (Figure 2
). 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 DMBAdA (22%) and DMBAdG (66%) adducts determined in vivo. This observation suggests that DMBAdA and DMBAdG 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, 2 test) or G:C (P = 0.1,
2 test) mutations (Table V
). 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:CT: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 VI
). 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 DMBAdG and DMBAdA 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:TT: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 SpragueDawley (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,
2 test; only 8/18 A:T
T:A mutations had the mutated dA 3' to a dC; Table V
) 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 V). 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 AT 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 DMBAdG and DMBAdA 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.
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Notes |
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