Time Course of cII Gene Mutant Manifestation in the Liver, Spleen, and Bone Marrow of N-Ethyl-N-Nitrosourea-Treated Big Blue Transgenic Mice

Jianyong Wang*,{dagger}, Xiaoli Liu*, Robert H. Heflich*,{dagger} and Tao Chen*,1

* Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research/FDA, Jefferson, Arkansas 72079; and {dagger} Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205

Received May 26, 2004; accepted July 26, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The time between treatment and the appearance of mutants (mutant manifestation time) is a critical variable for in vivo transgenic mutation assays. There are, however, limited data describing the optimal sampling time for detecting mutations in various tissues of mutagen-treated animals. In this study, we investigated the time course of cII gene mutant induction in the liver, spleen, and bone marrow of Big Blue transgenic mice treated with N-ethyl-N-nitrosourea (ENU). Six-month-old female mice were treated with a single dose (120 mg/kg) of ENU, and the animals were sacrificed, and the cII mutant frequencies (MFs) were determined at 1, 3, 7, 15, 30, and 120 days after the treatment. The MFs in the liver cII gene of ENU-treated mice increased with time after the treatment, while the MFs for concurrent controls remained constant. The liver cII MFs in ENU-treated mice were significantly increased at day 30 and 120 (p < 0.01), with the largest increase at day 120. The spleen cII MFs in ENU-treated mice were increased significantly at day 7 and later (p < 0.01), and reached a plateau at day 30. In the bone marrow, the cII MFs in ENU-treated mice were increased significantly at all sampling times (p < 0.01), with the maximum MF at day 3. These results confirm that the time after treatment required to reach the maximum MF is tissue specific, with the approximate time for the maximum ENU-induced cII MF response being: bone marrow, 3 days; spleen, 14–30 days; and liver, more than 30 days.

Key Words: Big Blue transgenic mice; N-ethyl-N-nitrosourea; mutation assay; mutant manifestation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transgenic animal mutation assays are reliable tools for detecting the mutagenicity of chemicals in vivo (Thybaud et al., 2003Go). Unlike in vitro methods, responses in these assays reflect the absorption, distribution, metabolism, and elimination processes in intact animals. There are at least four assays (lacI, lacZ, cII, and gpt-delta) that are generally accepted (Thybaud et al., 2003Go). Among the four target genes, the cII gene is the smallest (294 bp), which makes sequencing mutants very cost-effective in comparison with the larger lacI (1 kb) and lacZ (3 kb) genes. As part of the {lambda} phage genome, the cII gene can be used for mutation analysis with the two most common transgenic rodent models, MutaTMMouse and Big BlueTM rodents. Consequently, the cII mutation assay can be used to compare the results between the two models. Because the cII assay has the advantage of positive mutant selection and yields similar results to those obtained using the original lacI and lacZ assays, the cII assay is a promising labor-saving alternative for measuring in vivo mutation (Jakubczak et al., 1996Go; Swiger et al., 1999Go, 2001Go; Zimmer et al., 1999Go).

Transgenic mutation assays provide an opportunity to measure mutations in virtually any tissue. However, because different tissues have different turnover rates, drug metabolism, and DNA-repair capacities, it is presumed that expression time or mutant manifestation time in response to mutagen exposure also will differ. This makes sampling time a critical factor in the detection of chemical-induced mutagenicity (Heddle, 1999Go; Thybaud et al., 2003Go). Due to the high cost of conducting transgenic mutation assays, the protocol should be carefully designed to reduce the sample size. A simple and cost-effective protocol was established several years ago, in which different tissues were sampled at a single time when it was presumed that the mutant frequencies (MFs) in different tissues would have reached a plateau. This approach uses a minimum of animals, but it may lead to the underestimation of mutagenic potential, because the MF at the plateau may not be the maximum (Heddle et al., 2003Go).

Several studies have been conducted to investigate the time course of MF induction by different mutagens in different tissues, almost all using the lacZ transgene. Most of these studies, however, did not use sampling times before posttreatment day 7 or after posttreatment day 28 (Douglas et al., 1995Go, 1996Go; Hachiya et al., 1999Go; Hara et al., 1999Go; Mientjes et al., 1998Go; Sun et al., 1999Go; Suzuki et al., 1999Go; Thybaud et al., 2003Go). In addition, there is almost no mutant manifestation data for the cII gene. Therefore, in this study we have evaluated the in vivo mutant manifestation by treating Big Blue mice with a single dose of N-ethyl-N-nitrosourea (ENU) and measuring cII gene MF in liver, spleen, and bone marrow from 1 to 120 days after treatment.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animal treatment. The recommendations set forth by our Institutional Animal Care and Use Committee for the handling, maintenance, treatment, and sacrifice of animals were followed. Big Blue transgenic mice were purchased from Stratagene (La Jolla, CA). Six-month-old female mice were injected intraperitoneally with a single dose of 120 mg/kg body weight ENU (CAS# 759–73–9, Sigma, St. Louis, MO) or the vehicle dimethylsulfoxide (DMSO, Sigma) in a volume of 2 ml/kg body weight. Previous studies indicate that this dose produces a strong mutagenic response in multiple tissues (Zimmer et al., 1999Go). Groups of 4–5 animals were sacrificed by CO2 asphyxiation on posttreatment days 1, 3, 7, 15, 30, and 120, and liver, spleen and bone marrow were collected. These three tissues have slow, moderate, and fast turnover rates, respectively, and are tissues commonly used for in vivo MF analysis (Thybaud et al., 2003Go). The tissues were frozen immediately in liquid nitrogen and then stored at – 80°C.

DNA isolation. High-molecular-weight genomic DNA was extracted from the tissues using the RecoverEase® DNA isolation Kit (Stratagene) and following the manufacturer's instructions. DNA was dialyzed for 48 h with Tris-EDTA buffer, pH 7.5 (TE). The DNA recovered from the dialysis membrane was stored at 4°C.

cII Mutation assay. The procedures for the packaging of {lambda} phage, plating the packaged DNA samples, and the calculation of MFs were described in Chen et al. (2002)Go. Briefly, the {lambda} shuttle vector containing the cII target gene was rescued from genomic DNA with {lambda} phage packaging extract (Transpack; Stratagene). Then E. coli G1250 was mixed with 1:3000 dilutions of phage, plated on TB1 (tryptone-vitamin B1) plates, and incubated overnight at 37°C (nonselective conditions) to determine the total titer of packaged phages. For mutant selection, the packaged phages were mixed with E. coli G1250, plated on TB1 plates, and incubated at 24°C for 42 h (conditions for {lambda} cII selection). After incubation at 24°C, {lambda} phages with wild-type cII genes undergo lysogenization and become part of the developing bacterial lawn, whereas phages with mutated cII genes undergo lytic growth and give rise to plaques. When incubated at 37°C, {lambda} phages with both mutated and wild-type cII genes undergo a lytic cycle and result in plaque formation. The cII gene MF was the ratio of the total number of mutant plaques (as determined at 24°C) to the total number of plaques screened (as determined at 37°C).

Statistical analysis. One-way ANOVA followed by the Tukey test was used to evaluate the MF differences among groups.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MFs in Liver (Table 1 and Figure 1)
Compared with the controls, the cII MFs of the ENU-treated mice were significantly increased at days 30 and 120 after treatment (p < 0.01). ENU-induced MFs increased with time after the treatment, with the highest MF being measured at day 120 (5.3-fold over the controls). The ENU-induced MF at posttreatment day 120 also was significantly higher than at day 30 (p < 0.01).


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TABLE 1 cII Gene Mutant Frequencies (Mfs) in the Liver, Spleen, and Bone Marrow of Big Blue Mice

 


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FIG. 1. Time course of cII gene mutant frequency manifestation in the liver, spleen and bone marrow of Big Blue mice treated with 120 mg/kg ENUor DMSO. Data are taken from Table 1. Error bars equal one standard error.

 
MFs in Spleen (Table 1 and Figure 1)
Compared with the controls, the cII MFs of the ENU-treated animals were increased significantly at day 7 after treatment (p < 0.01) and gradually increased until a maximum was reached at day 30 (5.4-fold over the controls). There was no significant difference between the ENU-induced MFs at day 30 and day 120 after treatment. Also, the ENU-induced MFs at days 30 and 120 after treatment were significantly higher than those at days 1, 3, 7 and 15 (p < 0.05 for day 15 MF versus day 120 MF; p < 0.01 for all other comparisons), and the MF at day 15 was significantly higher than at day 7 (p < 0.01).

MFs in Bone Marrow (Table 1 and Figure 1)
Compared with the controls, the MFs of the ENU-treated mice were significantly increased at all sampling times (p < 0.01), reaching a maximum at day 3 (11.0-fold over the controls). Then the MFs gradually declined until a plateau was reached at day 15 (6.9-fold over the controls). The MF at day 3 was significantly higher than the MFs at all other sampling times (p < 0.05 for day 3 MF versus day 7 MF; p < 0.01 for all other comparisons). There was no significant difference among the ENU-induced MFs at days 1, 7, 15, 30, and 120 after the treatment.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sampling time is one of the most important factors for the measurement of mutations induced in vivo. Compared with in vitro assays, such as the Ames test (bacteria) or the Mouse Lymphoma assay (cultured cells), mutant manifestation processes for in vivo assays are complex. Various studies indicate that mutant manifestation is tissue specific (Hachiya et al., 1999Go; Hara et al., 1999Go; Sun and Heddle, 1999Go; Suzuki et al., 1999Go). There are many factors that contribute to this complexity, including mutagen pharmacokinetic characteristics (mutagen absorption, distribution, metabolism, and elimination), DNA repair, and tissue turnover rates. Suzuki et al. (1999)Go investigated the mutant manifestation of five mutagens and found that the results, even in the same organ, can be quite different. To reduce the influence of pharmacokinetic factors, ENU was selected for this study because it is direct acting, has a short half-life, and is evenly distributed to almost every tissue (Sun and Heddle, 1999Go).

Tissue turnover rate has been suggested as playing a major role in the distinct mutant manifestation patterns in different tissues. In this study, the liver cII MFs were not significantly elevated until posttreatment day 30 and increased slowly throughout the sampling period. We do not know whether the MF reached a maximum or not, since we did not sample at time points later than 120 days. Therefore, the time for maximum manifestation of liver cII mutants is greater than 30 days. In spleen, the ENU-induced cII MF reached a maximum, which was also the plateau level, on posttreatment day 30. Therefore, the mutant manifestation time for spleen was 14–30 days. The cII MF in bone marrow rose significantly with only one day's expression and reached a maximum at day 3. Then the MF decreased to a plateau level by day 15. These mutant manifestation results are consistent with the different turnover rates of the three tissues. The tissue turnover times of mouse bone marrow, spleen, and liver are approximately 2–3, 14–21, and 480–620 days, respectively (Cameron, 1971Go).

Unlike the ENU-induced MF time course in the spleen, the cII MF in bone marrow did not plateau at the maximum MF. Instead, the MF decreased after day 3 and reached a plateau on day 15. Studies of mutant manifestation in the lacZ gene produced similar results (Mientjes et al., 1998Go). These results indicate that the nature of the target cell population also plays an important role in the in vivo mutant manifestation process. In somatic tissues having some degree of cell replication, cells can be divided roughly into three populations: stem cells, transit cells, and nonreplicating differentiated cells. The DNA extracted for mutation assays comes from a mixture of the different cell populations, and the resultant MF is a weighted average of the actual MFs of the different cell populations (Heddle et al., 2003Go). Stem cells are regarded as the most important cell population in mutation assays and risk assesment (Heddle, 1999Go; Heddle and Swiger, 1996Go). Their continuous proliferation increases the risk for oncogenic mutation, while the differentiated cells, which are reproductively dead, cannot fix mutations and will eventually be shed from the tissue. Stem cells are responsible for the ultimate MF plateau, because eventually all the cells in the tissue will be replaced by the progeny of stem cells due to continuous tissue turnover. However, stem cells are a minority of the cells in animal tissues. Even in tissues with high turnover rates, such as bone marrow and intestinal epithelium, stem cells are less than 1% of the cell population (Heddle, 1999Go).

Transit cells, the other dividing cell population, are also important in mutant manifestation. Compared with stem cells, transit cells can make up a larger proportion of the cell population and also can divide more quickly than stem cells. The rapid cell division may shorten the time interval for DNA repair, and less DNA repair will result in higher induced MFs in transit cells. This may explain the MF peak seen in the time course of mutant manifestation in bone marrow. The decline in MF after day 3 is consistent with the replacement of mutated transit cells with the progeny of less mutated stem cells (Heddle et al., 2003Go). In addition, the MF plateau in spleen was very similar to that in bone marrow, and the time that the plateau level was reached in spleen was after that in bone marrow. A previous study on the induction of Hprt gene mutations in mice indicated that mutants measured in splenic T-lymphocytes were more likely to have been fixed in bone marrow than in spleen (Jansen et al., 1996Go). Our results also are consistent with the cII mutants in spleen resulting from mutations fixed in the stem cells of the bone marrow.

Hara et al. (1999)Go also observed a peak in N-propyl-N-nitrosourea-induced lacZ MF in bone marrow, followed by a decrease in MF, but the MF decrease continued after posttreatment day 14. Due to the limited number of sampling points in this study, it is not clear if the MF on day 28 was at a plateau or not. Their time course of mutant manifestation in the spleen was also different from what we observed. The MF in the lacZ gene reached a plateau by posttreatment day 7. Hara et al., however, used male mice that were much younger than ours (8–9 weeks vs. 24 weeks), and the age factor may have contributed to the difference in results. But the gender difference was suggested to have no significant effect on both spontaneous and ENU-induced mutation (Tao et al., 1993Go). Walker et al. (1999)Go studied the effects of age on Hprt mutant manifestation in the spleen of ENU-treated mice and observed that the rate of increase in ENU-induced mutant cells was inversely related to age; the older the animal at the time of ENU treatment, the longer the time span required to achieve the maximum MF. Younger animals may have had a larger transit cell population in bone marrow and a faster tissue turnover rate in spleen. These differences may partly explain the longer MF decrease in bone marrow and faster MF elevation in spleen observed by Hara et al. In addition, the different target genes used in the Hara et al. study and ours may also have contributed to the differences in the results.

In summary, our data suggest that the time required to reach the maximum MF in the cII gene of ENU-treated mice is tissue specific and depends upon the tissue turnover rate. In fast-dividing tissues, like bone marrow, the maximum MF occurs soon after treatment, and the maximum MF may be significantly different from the plateau level. These differences in mutant manifestation in different tissues indicate that an optimal sampling time is a very important factor in the establishment of rational protocols for transgenic mutation assays.


    ACKNOWLEDGMENTS
 
We thank the Oak Ridge Institute for Science and Education for partial funding of the project. We would also like to thank Drs. Anane Aidoo and Barbara L. Parsons for their review of this manuscript and their enlightening comments.


    NOTES
 
The views presented in this article do not necessarily reflect those of the Food and Drug Administration.

1 To whom correspondence should be addressed at HFT-130, NCTR, 3900 NCTR Rd., Jefferson, AR 72079, Fax: (870) 543-7682. E-mail: tchen{at}nctr.fda.gov.


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