N-Ethyl-N-nitrosourea (ENU) Increased Brain Mutations in Prenatal and Neonatal Mice but Not in the Adults

William Slikker, III*, Nan Mei{dagger} and Tao Chen{dagger},1

* College of Letters and Science, University of California, Los Angeles, California 90024, {dagger} Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research/US Food and Drug Administration, Jefferson, Arkansas 72079

Received March 9, 2004; accepted May 12, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The incidence of childhood cancer is increasing. One of the most common cancers for children under 15 years of age, gliomas for example, has been reported to have increased in incidence over the last 20 years by approximately 40%. The rising trend of childhood cancer in brain may be associated with environmental exposure to genotoxins and susceptibility to mutation in early development. To investigate age-dependent mutagenic sensitivity of brain tissue to genotoxins, the Big Blue mouse model was utilized in this study. Groups of five male mice were treated with a single dose of 120 mg/kg ENU transplacentally at three days before birth (prenatal), eight days (neonate) or eighteen weeks (adult) after birth. The animals were sacrificed six weeks after the treatment. The mutant frequencies and types of mutations in the brain cII gene from ENU-treated and concurrent control mice were determined. A significant increase in mutant frequencies over control was found in the prenatal and neonatal groups whereas there was no significant difference between the adult group and its control. Molecular analysis of the mutants also indicated that the mutational spectra from the ENU-treated mice were age-dependent. The percentage of A:T->T:A transversion, the typical type of mutation induced by ENU, was inversely related to the treatment age, whereas G:C->A: T transition was the main type of mutation in the adult group, the same as the control. These results demonstrate a differential mutagenic effect of ENU on the mouse brain depending on the stages of development and suggest an enhanced susceptibility of brain cancer hazard for perinatal exposure to genotoxicants.

Key Words: ethylnitrosourea; mutagenesis; carcinogenesis; development; Big Blue mouse.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The prevalence of childhood cancer and tumor development has significantly risen over the past few decades, increasing at roughly one percent per year, and the problem does not show signs of subsiding (Gurney et al., 1996Go, 1997Go; Kenney et al., 1998Go; Ries et al., 1997Go). An especially prominent increase in childhood cancer has been noted in the central nervous system, which is up 25% since the 1970's (Linet et al. 1999Go). The incidence of gliomas, for example, rose by about 40% for children in the under-15 age group over the last 20 years (Ries et al., 1999Go). Cancer of the central nervous system comprises 17% of all childhood cancers, making it the second most common kind of malignancy in childhood (Ries et al., 1999Go). Taking into account these recent trends in childhood brain cancer, it is important to ascertain if and why children are more susceptible to cancer so that we may begin to understand how to combat the current increases in childhood cancer.

Mutations are thought to be involved in the etiology of cancer because multiple genetic events are involved in every stage of carcinogenesis (Hennings et al., 1993Go; Loeb, 2001Go). It has been found that perinatal mice are highly sensitive to those carcinogens that exert tumorigenicity through a genotoxic mechanism (Dooley et al., 1988Go; Flammang et al., 1997Go; Fu et al., 1996Go). Therefore, it is reasonable to suspect that these animals also possess higher mutagenic sensitivity to genotoxins than adult animals. However, studies on age-related chemical mutagenicity are rare due to technical difficulty in measuring mutations in specific tissues from which tumors develop.

Big Blue transgenic mutation models represent a novel approach for studying mutant frequencies and types of mutations in nearly all tissues, thus permitting the identification of mutagenicity in specific tissues (Kohler et al., 1991aGo,bGo; Provost et al., 1993Go). In these systems, the chromosomally-integrated {lambda}LIZ/lacI or cII gene is used as the target for mutation. In this in vivo model, reporter genes are located on shuttle vectors that are derivatives of the bacteriophage lambda ({lambda}). Multiple genomic copies of the phage are contained within the genome of each transgenic animal cell as stably integrated concatamers. After exposure of transgenic animals to a test substance, DNA can be isolated from individual organs, and single copies of the phage genome can be excised from high molecular weight DNA and packaged into infectious particles with the help of a packaging mix (Kohler et al., 1990Go). If appropriate E. coli host cells are infected, plated, and incubated, plaques become visible on the plates within hours. Mutants can be selected and identified for mutations in several target genes (lacI, cI, and cII).

Rodents have long been used as animal models to predict and estimate the risk or effects associated with mutagens and carcinogens in the human population. The testing of mice with the mutagen N-ethyl-N-nitrosourea (ENU) serves a similar purpose in this study to predict the induced effects of a mutagen at differing stages in mammalian development. ENU is known as a potent mouse transplacental mutagen that works largely as an ethylating agent of DNA and has a LD50 in vivo of 240 mg/kg (Donovan and Smith, 1999Go; Heyting et al., 1980Go; Malling et al., 1999Go). In this study, Big Blue transgenic mice were administered a single dose of 120 mg/kg ENU at the time-points of three days before birth (prenatal), eight days after birth (neonatal), and eighteen weeks after birth (adult) and sacrificed six weeks after administration of ENU, along with corresponding controls. This choice of time-points enables a comparison of ENU's effects over the entire development of the rodent from prenatal to adult. The purpose of this study was to determine whether mutant frequencies and mutation types induced by ENU were associated with treatment age. The possible mechanisms of age-dependent mutation induction were also discussed.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals and treatments. Pregnant female Big Blue C57BL/6 transgenic mice were purchased from Taconic Laboratories (Germantown, NY). These mice had been bred with nontransgenic C3H male mice by the supplier. The B6C3F1 offspring were separated according to sex, pooled, and then distributed to individual litters during the first week following birth. Six to eight same-sex pups were assigned to each foster mother. The recommendations set forth by our Institutional Animal Care and Use Committee for the handling, maintenance, treatment, and sacrifice of the animals were followed. The ENU was purchased from Sigma (St. Louis, MO) and administered in dimethylsulfoxide (DMSO).

Animals were given a single ip injection of 120 mg ENU/kg body weight or vehicle control in a volume of 2 ml/kg body weight at various times during their development. For the prenatal treatment groups, five pregnant mice were treated at 18 days of gestation, and at birth the pups were pooled and assigned randomly to the females. For the postnatal treatment groups, the pups were first pooled and distributed randomly into the different groups at birth. Five male mice per treatment group received treatment at postnatal days (PNDs) 8 (neonatal group) or 126 (adult group). There were two concurrent controls. Mice were administered DMSO at PND 8 to serve as a control for the perinatal treatment groups and at PND 126 to serve as a control for the adult treatment group. The animals were sacrificed six weeks after their treatment. The brains were isolated, frozen quickly by using liquid nitrogen, and stored at –80°C.

Isolation of DNA from brain tissue. RecoverEase DNA isolation kit (Stratagene, La Jolla, CA) was used for the isolation. Each DNA sample was isolated from ~100 mg (about one quarter) of mouse brain tissue. Brain tissue was gently homogenized in 5 ml cold lysis buffer to disaggregate, and then sent through a sterile cell strainer into a 50 ml conical tube to be centrifuged at 1100 x g for 12 min at 4°C. The supernatant was discarded and a fresh 10 ml of cold lysis buffer was added for another centrifugation at 1100 x g for 5 min. at 4°C. The supernatant was again discarded and the residual droplets were removed with a sterile applicator. A digestion solution was composed of a 20 µl RNace-It ribonuclease cocktail in 1 ml digestion buffer. Seventy µl of this digestion solution and 70 µl of warmed proteinase K solution was mixed and added into the pellet. The conical tube was then placed in a 50°C water bath for 45 min. The genomic DNA was then transferred to a dialysis cup floating on Tris-EDTA(TE) buffer prepared in advance, and left to dialyze at room temperature for approximately 25 h. The fully hydrated genomic DNA was then removed from the dialysis cup and stored at 4°C until use.

cII mutation assay. The packaging of the phage, plating of the packaged DNA samples, and determination of mutant frequency (MF) were carried out following Stratagene's procedure for the {lambda} select-cII mutation detection system for Big Blue rodents. The shuttle vector containing the cII target gene was rescued from total genomic DNA with phage packaging extract (Transpack; Stratagene). The plating was performed with the Escherichia coli host strain G1250. The bacteria were grown in TB1 liquid medium with 1% maltose-MgSO4 (1 M) overnight at 30°C in preparation for the experiment. To determine the total titer of packaged phages, G1250 bacteria were mixed with a 1:3000 dilution of phage, plated on TB1 plates, and incubated overnight at 37°C (nonselective conditions). For mutant selection, the packaged phages were mixed with G1250, plated on TB1 plates and incubated at 24°C for about 42 h (conditions for cII selection). The cII MF was calculated as 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).

Sequence analysis of the cII mutants. The sequencing protocol was adapted from a previous procedure with minor modifications (Chen et al., 2002Go). The cII mutant plaques were selected at random from different animals and replated at low density to verify the mutant phenotype. Single, well-isolated plaques were selected from these plates and transferred to a microcentrifuge tube containing 50 µl of autoclaved distilled water. The tubes were placed in a thermocycler at 99.9°C for 5 min and centrifuged at 1500 x g for 5 min immediately after the heating. The cII target DNA was amplified by PCR with primers 5'-AAAAAGGGCATCAAATTAACC-3' and 5'-CCGAAGTTGAGTATTTTTGCTG-3'. For PCR amplification, 10 µl of the supernatant and 0.03 µl of the each primer (148 µM) were added to 10 µl of 2x PCR Master Mix (Promega, Madison, WI). The PCR reaction was carried out with the following cycling parameters: a 3 min denaturation at 95°C; followed by 30 cycles of 30 s at 95°C, 1 min at 60°C, and 1 min at 72°C; with a final extension of 10 min at 72°C. The PCR products were purified using PCR purification kits (Qiagen, Chatsworth, CA). The cII mutant DNA was sequenced and analyzed with a CEQ DTCS-Quick Start Kit and a CEQ 8000 Genetic Analysis System (Beckman Coulter, Fullerton, CA) according to the manufacturer's instructions. The primers for cII mutation sequencing were the same as those used for the PCR.

Statistical analyses. Statistical analyses of MFs were performed using SigmaStat (SPSS Science, Chicago, IL). All MF data are expressed as the mean ± standard deviation (SD) from five different animals. Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Student–Newman–Keuls test for comparison of multiple treatment groups. Because the variance increased with the magnitude of the MF, the data were log-transformed before conducting the analysis. The difference of mutational types between different groups was paired and tested statistically using the computer program written by Cariello et al. (1994)Go for the Monte Carlo analysis developed by Adams and Skopek (1987)Go.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mutant Frequency
The sample groups containing five male Big Blue mice were administered 120 mg/kg ENU at different time-points and sacrificed six weeks after administration with corresponding controls. The raw data for the brain cII MFs from ENU-treated or control Big Blue mice are shown in Table 1 and summarized in Figure 1. The mice treated with ENU perinatally showed a significant increase in MF when compared to their concurrent controls, with an increase of approximately 6-fold for prenatal treatment (p < 0.001) and 2.3-fold for neonatal treatment (p <0.05). In addition, these test groups showed a significant increase in their MFs when compared to the adult treatment group (prenatal vs. adult, p<0.001 and neonate vs. adult, p < 0.01). There is also a significant difference between the prenatal and neonatal treatment groups (p < 0.001). The adult treatment group, however, did not show any significant difference from controls (p = 0.25).


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TABLE 1 Brain cII Mutant Frequencies from ENU-Treated or Control Big Blue Male Mice

 


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FIG. 1. Mutant frequencies (MFs) at different treatment time points in the brain cII gene from male Big Blue mice treated with 120 mg ENU/kg body weight and sacrificed six weeks after the treatment. PND–3 is the time point when the animals were treated transplacentally three days before birth. Each data point represents the average MFs from five animals ± standard deviation. ENU significantly increased the MFs over the controls at prenatal (PND–3) (p < 0.001) and neonatal (PND8) (p < 0.01) mice while no significant increase was found between the adult treatment (PND126) and its concurrent control (p = 0.25). The ENU-induced MFs from prenatal mice were significantly higher than those from neonatal mice and adult groups (p < 0.001, respectively). Also, the MFs from neonatal treatment were higher than those from adult treatment (p < 0.05).

 
Mutation Types
ENU-induced brain cII mutations were evaluated by DNA sequence analysis of 51 prenatal, 51 neonatal, 41 adult mutants isolated from the ENU-treated sample groups, as well as 38 control mutants. Mutations that were found more than once in the mutants isolated from a single animal were assumed to be siblings and were considered to represent a single independent mutation. Accordingly, a total of 46 independent mutations from prenatal, 47 from neonate, 39 from adult and 34 from control mice were identified (Table 2). Table 3 summarizes the types of mutations in the brain cII gene from the ENU-treated prenatal, neonatal, and adult mice as well as the control male mice. A statistical pairwise multiple comparison of these mutational spectra are listed in Table 4. Among the independent mutations of the prenatal and neonatal treatment groups, base pair substitutions were the most common type of mutation, with the A:T->T:A transversion being the most common type of base substitution. The adult and control groups likewise had base substitutions as their most common type of mutation. However, the most common type of base substitution for them was the G:C->A:T transition. There was no statistically significant difference between prenatal and neonatal treatment groups (p = 0.631) or between adult and control groups (p = 0.392). There were significant differences for several paired comparisons: prenatal treatment group vs. adult treatment group (p = 0.002), prenatal treatment group vs. control group (p = 0.001), neonatal treatment group vs. adult treatment group (p = 0.024), and neonatal treatment group vs. control group (p = 0.014).


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TABLE 2 Mutations in the Brain cII Gene from N-Ethyl-N-nitrosourea-Treated Prenatal, Neonatal, Adult, and Control Big Blue Mice

 

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TABLE 3 Summary of the Independent Mutations in the Brain cII Gene from N-Ethyl-N-nitrosourea-Treated Prenatal, Neonatal, Adult, and Control Big Blue Mice

 

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TABLE 4 Statistical Pairwise Multiple Comparison of Mutational Spectra in the Brain cII Gene from N-Ethyl-N-nitrosourea-Treated Prenatal, Neonatal, Adult, and Control Big Blue Mice

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ENU is a potent direct-acting alkylating agent that has been found to be mutagenic in a wide variety of mutagenicity test systems and carcinogenic in various organs of mammals (Shibuya and Morimoto, 1993Go). It has been shown to act as a neurocarcinogen in experimental studies with mice and other animals (Druckrey et al., 1973Go; Rice and Ward, 1982Go). ENU induces neoplasms that have morphological and biological similarities to naturally occurring neural tumors in human and animals and it has a referential transplacental and neonatal action, a single iv or ip injection inducing an almost 100% incidence of neural neoplasms (Lantos, 1986Go). Therefore, ENU is an ideal experimental model for the study of mutagenicity of brain tissue. Also, the mutagenic response to ENU should not depend on possible differences in the metabolic capabilities of animals of different ages due to its direct action. The animals were given 120 mg/kg ENU, which is one-half the LD50 ip, to ensure that mutations would arise and any differences in MF due to in the animal's maturity could be determined.

When ENU was administered perinatally, a significant increase in MF was seen when compared to the adult treatment group and to the controls, whereas the adult treatment group showed no significant increase of MF. The younger the animals when treated with ENU, the higher the MF. Mientjes and his colleagues (1996, 1998) have treated adult lacZ mice with ENU and observed no MF increase in the brain of the ENU-treated mice although O6- and N7-DNA adducts were present. This finding is consistent with our data from ENU-treated adult Big Blue mice. These data supports the hypothesis that younger mice are more sensitive to insults of mutagens than adults.

Analysis of mutant spectra in the brain cII gene from the different groups further confirms that ENU increased brain mutations in prenatal and neonatal mice but not in the adults. The main type of mutation G:C->A:T transition in the control and adult groups (44 and 56% for control and adult vs. 26 and 28% for prenatal and neonate) has been shifted to A:T->T:A transversion and A:T->G:C transition in the perinatal treatment groups (47 and 38% for prenatal and neonate vs. 10 and 6% for adult and control). Also a higher percentage of G:C->A:T transition from the adult and control treatment groups occurred at CpG site than those from the prenatal and neonatal treatment groups (68 and 87% for adult and control vs. 50 and 54% for prenatal and neonate). These types of mutations are consistent with DNA adduct formation induced by ENU. ENU has been shown to react with oxygen in DNA and result in modification at the O6 position of guanine and at the O4 and O2 positions of thymine. O6-ethylguanine that induces G:C->A:T transition, can be efficiently repaired by O6-ethylguanine-DNA-alkyltransferase in normal mammalian systems. Therefore, A:T->T:A transversion induced by O2-ethylthymine and A:T->G:C transition induced by O4-ethylthymine become the major types of mutation because O2- and O4-ethylthymine adducts are persistent in normal mammalian cells after ENU treatment (Bronstein et al., 1992Go; Jansen et al., 1994Go, 1995Go; Liem et al., 1994Go). Our results also indicate that spontaneous and ENU-induced types of mutations in the mouse brain are similar to those in other mouse tissues (Hill et al., 2004Go; Mittelstaedt et al., 1995Go; Walker et al., 1996Go).

The mechanisms for the age-related sensitivity to mutagenicity are still unknown. It has been reported that the penetration of ENU into brain is not affected by the brain blood barrier, and that DNA alkylation products are readily formed in the brain tissue (Branstetter et al., 1987Go). Therefore, distribution of ENU in brain is not the main factor that causes the susceptibility at perinatal developmental stages. Cell proliferation, however, has been considered a main factor for determining susceptibility at different developmental stages. High rates of cell proliferation can increase the probability that an initiated clone will expand before cell death (Ginsberg, 2003Go). We sequenced the cII DNA of mutants from prenatal, neonatal, and adult mice treated with ENU, and vehicle control to observe if there was any difference among these mice due to clonal expansion. If an initially mutated clone was expanded and resulted in a higher MF, we would find a number of sibling mutants in an animal. Such events, however, were rare. About 10 mutants from each animal were analyzed. Forty-six of 51 mutations in ENU-treated prenatal mice, 47 of 51 mutations in ENU-treated neonates, 39 of 41 in ENU-treated adults and 34 of 38 in the control mice were independent. In all of these groups including the control, only about 5–10% mutants resulted from clonal expansion. Therefore, the sequencing data gave no evidence for clonal expansion playing a crucial role for increased mutant frequencies found in perinatal mice treated with ENU.

It has been postulated that cell proliferation might be requisite for both DNA repair and gene mutation in vitro (Bielas and Heddle, 2000Go) and for expression of chemical-induced mutagenicity in vivo (Cunningham and Matthews, 1995Go). Mutations are established generally more effectively in rapidly dividing tissues because the higher rate of cell division increases the number of cells in S phase, which tend to make the cell population more vulnerable to the genotoxic effects of chemicals and leaves less time for DNA repair prior to fixation of the damage as a mutation (Bertram and Heidelberger, 1974Go; Rabes et al., 1986Go; Stuart et al., 2000Go). Most of the cells in the fetal brain divide rapidly and the proliferative activity slows down in the neonate while they rarely divide in the adult (Korr, 1980Go). A mouse brain approaches maturity at 2–3 weeks of age (Kobayashi, 1963Go), and becomes practically mitotically quiescent (Korr, 1980Go). The brain cII MFs at different developmental stages appear related to the proliferative activity of brain cells. Our results suggest that rapid cell division in earlier stages of development is a determining factor for the greater mutagenic sensitivity of perinatal life.

While we do know that certain types of childhood cancers are increasing, we don't know why. We know little about how chemicals in the environment relate to risks of childhood cancer (Goldman, 1998Go). Animal models provide the opportunity for testing chemical tumorigenic potency and hypotheses that cannot be tested easily or ethically in humans. In this study, we found that the carcinogenic response of mouse brain tissue to ENU at different ages is correlated well with the mutagenic response. Brain tumors have been induced prenatally in offspring of ENU-treated pregnant female mice or by ENU treatment of neonatal mice. However, the treatment of adult mice with various carcinogens has not been effective in inducing brain tumors (Schmahl et al., 1990Go; Vesselinovitch et al., 1974Go). This correlation indicates that mutation plays an important role in the perinatal susceptibility to carcinogenesis of chemicals and suggests that the higher incidence of childhood cancer can be the result of the early life's vulnerability to genotoxic carcinogens existing in the environment.

Ginsberg (2003)Go recently provided a review of juvenile animal bioassay data in comparison to adult animal data for a broad array of carcinogens. He found that short-term exposures in early life were likely to yield a greater tumor response than short-term exposures in adults, but produced similar tumor response when compared to long-term exposures in adults. Therefore, he suggested that early life sensitivity should be incorporated into cancer risk assessment for children. Our results from this study appear to support his view because of the increase in mutagenic sensitivity to genotoxic carcinogens seen in the prenatal and neonatal mice and due to the known importance of mutations in carcinogenesis. The early life exposures should not simply be prorated over an entire life span for assessing cancer risks of genotoxic chemicals.

Although the cII gene in the Big Blue mouse is a transgene, the mutations that occur at this gene serve as a good indication of mutations that occur at other endogenous genes including oncogenes. The correlation between tumor incidences in mouse brain tissue and the MFs in the brain cII gene at different developmental stages further confirms that transgenic mutagenicity methods can serve as a short term assay to predict the carcinogenicity of genotoxic agents. In addition, the findings of this study provide certain applications and insights for future research on in vivo mutagenesis because the age at which a mutagen is administered has a significant effect on the outcome produced by the mutagen. Therefore, when designing a treatment schedule for a mutagenicity study, one should consider the age factor, especially during the perinatal stages of life, when the brain tissue is uniquely sensitive to chemical mutagenesis.


    ACKNOWLEDGMENTS
 
We thank the Oak Ridge Institute for Science and Education for partial funding of the project. We would also like to thank Drs. Mugimane G. Manjanatha and Carrie R. Valentine for their enlightening comments and hearty discussions in reviewing the data.


    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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