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2005 33: 307Toxicol Pathol
Hegi and Robert C. Sills
Yongbaek Kim, Hue-Hua L. Hong, Yan Lachat, Natasha P. Clayton, Theodora R. Devereux, Ronald L. Melnick, Monika E.
Genetic Alterations in Brain Tumors Following 1,3-Butadiene Exposure in B6C3F1 Mice
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Toxicologic Pathology, 33:307–312, 2005
Copyright C ?by the Society of Toxicologic Pathology
ISSN: 0192-6233 print / 1533-1601 online
Genetic Alterations in Brain Tumors Following 1,3-Butadiene
Exposure in B6C3F1 Mice
YONGBAEK KIM,1HUE-HUA L. HONG,1YAN LACHAT,2NATASHA P. CLAYTON,1THEODORA R. DEVEREUX,1
RONALD L. MELNICK,1MONIKA E. HEGI,2AND ROBERT C. SILLS1
1Laboratory of Experimental Pathology, National Institute of Environmental Health Sciences, Research Triangle Park,
North Carolina, USA
2Laboratory of Tumor Biology and Genetics, Department of Neurosurgery, University Hospital Lausanne, Lausanne, Switzerland
However, 6 malignant gliomas and 2 neuroblastomas were observed in B6C3F1 mice exposed to 625 ppm 1,3-butadiene (NTP technical reports 288
and 434). These mouse brain tumors were evaluated with regard to the profile of genetic alterations that are observed in human brain tumors.
Alterations in the p53 tumor suppressor gene were common. Missense mutations were observed in 3/6 malignant gliomas and 2/2 neuroblastomas
and were associated with loss of heterozygosity. Most of the mutations occurred in exons 5–8 of the p53 gene and were G → A transitions, and did
not involve CpG sites. Loss of heterozygosity at the Ink4a/Arf gene locus was observed in 5/5 malignant gliomas and 1/1 neuroblastoma, while the
PTEN (phosphatase and tensin homologue) gene locus was unaffected by deletions. One of 2 neuroblastomas had a mutation in codon 61 of H-ras,
while H-ras mutations were not observed in the malignant gliomas examined. Only 1 brain tumor has been reported from control mice of over 500
NTP studies. This malignant glioma showed no evidence of alterations in the p53 gene or K- and H-ras mutations. It is likely that the specific genetic
alterations observed were induced or selected for by 1,3-butadiene treatment that contributed to the development of mouse brain tumors. The observed
findings are similar in part to the genetic alterations reported in human brain tumors.
B6C3F1 mouse; 1,3-butadiene; brain tumors; p53; Ink4a/Arf; mutation; loss of heterozygosity; neurocarcinogenesis.
Brain tumors in humans are the third most common cause
of death among 18- to 35-year olds, and their incidence is in-
creasing (Kleihues and Cavenee, 2000). With the exception
of familial cancer syndromes (Li-Fraumeni) involving the
nervous system and irradiation, the etiology of brain tumors
is still largely unknown (Kleihues and Cavenee, 2000). Epi-
demiological studies have shown that increased risk of brain
tumor development is associated with certain occupations,
such as farming, dentistry, fire fighting, metal works, and the
rubber industry, but specific exposures or causative environ-
mental agents have not been identified (Thomas et al., 1986;
Kaplan et al., 1997). Malignant glioma is the most common
primary malignant brain tumor in adults, while neuroblas-
(DeAngelis, 2001). Malignant gliomas appear to develop by
a stepwise accumulation of genetic lesions (Holland, 2001;
Schwab et al., 2003). Genetic alterations frequently detected
in human gliomas, include inactivation of tumor suppressor
genes such as TP53 (Rasheed et al., 1994), retinoblastoma
(RB) (He et al., 1995), Ink4a/Arf (He et al., 1995), PTEN
Address correspondence to: Dr. Robert C. Sills, National Institute of
Triangle Park, NC 27709, USA; e-mail: email@example.com
Abbreviations: DNA: Deoxyribonucleic acid; dNTP: Deoxynucleotide;
EGFR: Epidermal growth factor receptor; LOH: Loss of heterozygosity;
Polymerase chain reaction; PTEN: Phosphatase and tensin homologue.
(phosphatase and tensin homologue) (Li et al., 1997), acti-
vation of growth-promoting pathways e.g., amplification of
CDK4 (He et al., 1995), and amplification or mutation of the
epidermal growth factor receptor (EGFR) (Ekstrand et al.,
Human neuroblastomas exist as 3 nonoverlapping sub-
types exhibiting distinct genetic and morphological features
(Lastowska et al., 2001). Chromosome 1p deletion and 17q
gain represent early events in transformation, while N-myc
gene amplification is a late event contributing to tumor pro-
gression (Tonini and Romani, 2003).
Ten of nearly 500 National Toxicology Program (NTP)
studies resulted in equivocal neurocarcinogenic effects in the
F344 rat brain (Melnick and Huff, 1992; Sills et al., 1999).
been a target for chemical carcinogenesis in NTP bioassays
(Sills et al., 1999). Only 1 spontaneous malignant glioma has
been found in 1100 chamber control male mice in the NTP
studies (Haseman et al., 1999).
in the production of rubber and plastics, and is also gen-
erated during the combustion of fossil fuels and is thus
present in the environment (Himmelstein et al., 1997). 1,3-
smoke (Brunnemann et al., 1990). Inhalation exposure of
1,3-butadiene to Sprague–Dawley rats did not induce brain
tumors (Owen et al., 1987). However, the development of
malignant gliomas and neuroblastomas in B6C3F1 mice ex-
posed to 1,3-butadiene provided the first opportunity to com-
pare genetic alterations in chemically induced mouse brain
tumors with those of humans (NTP, 1984, 1993). Exposure
308KIM ET AL.
of mice with 1,3-butadiene also caused increased incidences
of tumors in multiple organ systems and mutations in tumor
suppressor genes and oncogenes were often detected (Hong
et al., 2000; Walker and Meng, 2000; Zhuang et al., 2000;
Sills et al., 2001; Zhuang et al., 2002).
2 NTP studies in which B6C3F1 mice were exposed to 1,3-
butadiene (NTP, 1984, 1993). Candidate genes commonly
altered in human brain tumors (p53, Ink4a/Arf, PTEN) were
analyzed. In addition, genetic alterations in the K-ras and
H-ras genes were examined. The results of this study will
provide further insights into neurocarcinogenesis, including
the potential role of environmental chemical exposure as a
causative agent in the development of human brain tumors.
MATERIALS AND METHODS
Male and female B6C3F1 mice, 6–8 week of ages, were
exposed to 200, 312, 625, or 1250 ppm 1,3-butadiene by in-
halation 6 hour/day, 5 days/week for 13, 26, 40, or 60 weeks
2 neuroblastomas were observed in treated mice (Table 1). A
total of 9 brain tumors (including 1 spontaneous tumor from
another NTP study) were fixed in 10% neutral buffered for-
malin, routinely processed, embedded in paraffin, sectioned
to a thickness of 5 µm, and stained with hematoxylin and
were prepared from each paraffin block containing the brain
neoplasms for isolation of DNA for PCR-based assays.
Immunohistochemistry on Paraffin Sections
After deparaffinization, sections were incubated for 20
minutes in methanol 2% H2O2, then rinsed 3 times in PBS
buffer. For antigen retrieval, sections were treated for 4 min-
utes in a pressure cooker using 10 mM citrate buffer (pH
6.0) then cooled in PBS for 20 minutes. The sections were
incubated with normal goat serum (1/20 in PBS) for 30 min-
utes in order to reduce nonspecific binding followed by ap-
plication of the primary antibody (NCL-p53-CM5p, Novo-
castra Laboratories Ltd, Newcastle, UK, diluted 1/500) at
room temperature for 60 minutes. Secondary antibodies with
standard incubations were performed using Vectastain Elite
Vector Rabbit kit (Ig-6101, Vector Lab Inc, Burlingam, CA,
USA). The chromogen used was diaminobenzidin tetrahy-
terstained with Gill’s hematoxylin for 30 seconds, rinsed 3
times in PBS for 5 minutes, dehydrated in ethanol xylene
gradients, and mounted using Eukitt solution.
TABLE 1.—Brain tumor incidences in B6C3F1 mice exposed to 1,3-butadiene.a
Malignant glioma0/50 TR 288
aGroups of 50 male mice were exposed to air containing 1,3-butadiene 6/h/d, 5d/wk for
indicated periods in parentheses and held for up to either 60 weeks (TR 288) or 104 weeks
bTR = National Toxicology Program Technical Report.
DNA Isolation, Amplification, and Cycle Sequencing
The methods for DNA isolation and sequencing have been
paraffin-embedded sections containing brain neoplasms us-
nested primers for K- and H-ras. Touch-down PCR was per-
primers and annealing temperature profile of the cycles have
been reported previously (Hong et al., 2000). Normal con-
trols for K- and H-ras, or p53genes and no DNA con-
trols were run with all sets of reactions. To identify muta-
tions, samples were sequenced utilizing a cycle sequencing
kit (U.S. Biochemical, Cleveland, OH), which incorporates
α-33P-dideoxynucleotide (ddNTP) terminators (A,C,G,T)
ucts were purified using a QIAquick Gel Extraction Kit
(QIAGEN, Valenicia, CA). The amplification primers also
served as sequencing primers. Mutation identification was
Analysis for Loss of Heterozygosity (LOH) in Mouse
embedded sections using a clean razor blade without taking
normal brain. Gliomas and neuroblastomas were digested
in 50 µl Tail Buffer (50 mM KCl, 10 mM Tris/HCl pH
9.0, 0.45% Nonidet P-40, 0.45% Tween 20) in the pres-
5 hours at 55◦C at 400 rpm. Proteinase K was inactivated by
heating samples for 15 minutes at 85◦C. Two µl of DNA
were used for LOH analysis performed by PCR. Reactions
50 seconds at 55◦C, 30 seconds at 72◦C), and 10 minutes
at 72◦C for final extension in a thermal cycler (GeneAmp
PCR 9700, Applied Biosystem, Foster City, CA). Markers
used for LOH analysis were D11Mit320 (ca 0.3 cM from
p53), D19Mit19 (ca 1.7 cM from PTEN), and D4Mit27 (ca
7.7 cM from Ink4a/Arf) ?http://www-genome.wi.mit.edu?;
Six malignant gliomas and 2 neuroblastomas from
B6C3F1 mice exposed to 1,3-butadiene were detected at
necropsy (NTP, 1984, 1993). All of the malignant gliomas
occurred in the anterior or olfactory lobe of the cerebrum
densely cellular masses and effaced the neuropil of the olfac-
tory lobe. Tumor cells invaded the adjacent parenchyma of
the cerebrum (Figure 1b). Malignant gliomas consisted of
pleomorphic and poorly differentiated glial cells with giant
tected in the olfactory lobe. The tumors were of high cellular
density and invaded the adjacent neuropil. Neoplastic cells
Vol. 33, No. 3, 2005
1,3-BUTADIENE MOUSE BRAIN TUMORS309
neoplastic tissue (arrow) in the olfactory lobe of the cerebrum. Bar=0.45 cm. (b)—Brain; male B6C3F1 mouse exposed to 625 ppm 1,3-butadiene for 26 weeks and
H&E. Bar=250 µm. (c)—Brain; male B6C3F1 mouse exposed to 625 ppm 1,3-butadiene for 26 weeks and held for 2 years. Malignant glioma. Neoplastic cells
were diffusely arranged, pleomorphic and poorly differentiated, and contained occasional giant nuclei (long arrow). Atypical mitotic figures (short arrow) were also
present. H&E. Bar=70 µm. (d)—Brain; male B6C3F1 mouse exposed to 625 ppm 1,3-butadiene for 13 weeks and held for 2 years. Neuroblastoma. Neoplastic
cells formed rosettes (asterisk) surrounding an eosinophilic center, or pseudorosettes around blood vessels (Bv). Neoplastic cells were small and monomorphic, and
have small round hypo- and hyperchromatic nuclei. Atypical mitotic figures (arrow) were present. H&E. Bar=40 µm. (e)—Brain; male B6C3F1 mouse exposed to
625 ppm 1,3-butadiene for 26 weeks and held for 2 years. Malignant glioma. Note the striking accumulation of the p53 protein (brown chromogen) in nuclei of the
neoplastic cells. Avidin-biotin peroxidase complex method for p53 protein with hematoxylin counterstain. Bar=70 µm.
arranged around cores of cytoplasmic fibrils or blood vessels
were occasionally observed in the tumors. The morphologic
characteristics of malignant gliomas and neuroblastomas are
consistent with those reported for humans (Kleihues and
Cavenee, 2000). With immunohistochemical staining, 3 of
5 malignant gliomas and both neuroblastomas exhibited nu-
clear accumulation of p53 protein (Figure 1e).
The 6 malignant gliomas and 2 neuroblastomas from
B6C3F1 mice exposed to 1,3-butadiene were analyzed for
310KIM ET AL.
TABLE 2.—Summary of genetic alterations in brain tumors from B6C3F1 mice exposed to 1,3 butadiene.a
(Exons 5, 6, 7, 8)
MG (78 wk)
MG (79 wk)
MG (95 wk)
MG (41 wk)
MG (60 wk)
MG (60 wk)
NB (49 wk)
NB (83 wk)
625 ppm (26 W)
625 ppm (13 W)
625 ppm (13 W)
625 ppm (60 W)
625 ppm (60 W)
1250 ppm (60 W)
625 ppm (13 W)
625 ppm (13 W)
Codon 263: GGA→AGA (Gly→Arg)
Codon 192: ATC→TTC (Ile→Phe)
Codon 170: GTG→ATG (Val→Met)
Codon 263: GGA→AGA (Gly→Arg)
Codon 132: TGC→TAC (Cys→Tyr),
Codon 263: GGA→AGA (Gly→Arg)
aND=not done, N=no mutation or no LOH, -B=Loss of C57BL/6 allele, -H=Loss of C3H allele, -pB=partial loss of C57BL/6 allele, -pH=partial loss of C3H allele.
bMG=malignant glioma, NB=neuroblastoma, In parenthesis=Age of animal when the tumors were diagnosed.
cGroups of 50 male mice were exposed to 1,3-butadiene 6/h/d, 5 d/wk for indicated periods in parentheses and held for up to either 60 weeks (TR 288) or 104 weeks (TR 434).
dNational Toxicology Program (NTP) technical reports.
fLOH=loss of heterozygosity, PTEN =phosphatase and tensin homologue genes.
gGln → Arg.
genetic alterations in the p53, K-, and H-ras genes. The pro-
file of genetic alterations is summarized in Table 2. Missense
gliomas and in both neuroblastomas. Mutations in p53 were
identified at codons 132, 170, 192, and 263 and consisted
mostly of G → A transitions (5/6) (Figure 2). Furthermore,
from B6C3F1 mice exposed to 1,3-butadiene. Sequencing panels a and c are
normal p53 exon 5 codon 132 and exon 8 codon 263, respectively, panels b
from brain tumors with mutated sequences at nucleotide indicated by arrows.
Note only the mutated allele is visible and the wild-type allele is lost.
LOH at the p53 gene locus was observed in 4 of 5 malignant
gliomas and both neuroblastomas. All of the tumors exhib-
ited loss of the C3H (H) allele with the exception of 1 tumor
with partial loss of the C57 (B) allele (Figure 3). LOH was
Ink4a/Arf. All of the malignant gliomas and neuroblastomas
gene locus. However, no LOH was observed near the PTEN
gene locus in any of the tumors examined. One neuroblas-
toma had a CAA → CGA mutation in codon 61 of the H-ras
gene, while no K-ras mutations were identified. In contrast
to the chemically induced brain tumors, the spontaneous ma-
lignant glioma was negative for mutations in K-ras, H-ras,
and exons 5–8 of the p53 gene. Likewise, no p53 protein ex-
pression was detectable in the spontaneous brain tumor by
FIGURE 3.—Loss of heterozygosity analysis of p53 gene in brain tumors
from B6C3F1 mice exposed to 1,3-butadiene. DNA was isolated from paraffin
embedded tumors and analyzed for loss of heterozygosity. The analysis was
products were loaded on a 2% agarose gel for separation of both alleles. Sample
C3H, and sample #4 (malignant glioma) had a partial loss of wild-type allele
C57BL/6. Positive control was obtained from genomic DNA of C57BL/6-C3H
mouse, water was used as negative control for the PCR reaction.
Vol. 33, No. 3, 2005
1,3-BUTADIENE MOUSE BRAIN TUMORS311
Despite the equivocal increase of brain tumors determined
by stastistical analysis, the location and specific genetic al-
terations observed in our study suggest that brain tumors in
mice exposed to 1,3-butadiene may be chemically induced.
In addition, the genetic alterations are similar to the major
molecular pathways observed in human brain tumors and
suggest a possible role of environmental exposure in human
neurocarcinogenesis. The predominant G → A transition in
the p53 gene in these mouse brain tumors suggests that treat-
ment of 1,3-butadiene may have caused DNA damage as has
et al., 1994; Zhuang et al., 1997; Hong et al., 2000; Sills
et al., 2001; Zhuang et al., 2002). The G → A transition ap-
lating agents, including 1,3-butadiene, that frequently cause
G:C → AT transition mutations, suggesting that they may
from cross linking of DNA strands (Trukhanova et al., 1998;
Melnick, 2002). Alternatively, transition mutations could be
spontaneous rather than as a consequence of interaction with
environmental carcinogens, as suggested in sporadic and fa-
milial human astrocytic tumors (Kleihues et al., 1995). In
some studies, up to 67% of human secondary glioblastomas
had TP53 mutations, and the majority of the mutations were
G → A transitions (Watanabe et al., 1996; Fulci et al., 2000).
All tumors evaluated for LOH in the vicinity of Ink4a/Arf
gene locus lost the C57BL/6 allele. The preference for loss
of the C57BL/6 allele of Ink4a/Arf is expected if p16Ink4ahas
of such brain tumors. The C3H allele of this gene is known
to code for a p16Ink4aprotein that has been shown to be de-
fective in its function to inhibit CDK4 due to 2 amino acid
changes in exon 2 (Herzog et al., 1999; Zhang et al., 2002).
Despite the small sample number, this observation supports
the notion that p16Ink4aplays a key role in the development
of mouse brain tumors as has been suggested in other studies
(Bachoo et al., 2002).
An increased risk of human brain tumor development has
been associated with certain occupations (Thomas et al.,
1986; Kaplan et al., 1997). However, specific causative en-
vironmental exposures with the exception of therapeutic X-
low rate of spontaneous brain tumors in B6C3F1 mice, the
effects of 1,3-butadiene in the present study were considered
equivocal, suggesting that the marginal increase in brain tu-
mors may be related to 1,3-butadiene treatment (Sills et al.,
1999). The mouse brain tumors examined were present in
the anterior or olfactory lobes and the genetic alterations ob-
served in the present study were most likely associated with
1,3-butadiene caused early genetic events in brain tumor in-
duction or simply accelerated the development of sponta-
neous brain lesions. However, the predominant pattern of
p53 transition mutations in mice exposed to 1,3-butadiene
for only 13 weeks suggests that p53 mutations may represent
an early event.
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