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2009 37: 835 originally published online 21 October 2009 Toxicol Pathol
Mark J. Hoenerhoff, Hue Hua Hong, Tai-vu Ton, Stephanie A. Lahousse and Robert C. Sills
National Toxicology Program Bioassays and Their Relevance to Human Cancer
A Review of the Molecular Mechanisms of Chemically Induced Neoplasia in Rat and Mouse Models in
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A Review of the Molecular Mechanisms of Chemically Induced
Neoplasia in Rat and Mouse Models in National Toxicology Program
Bioassays and Their Relevance to Human Cancer
MARK J. HOENERHOFF,1HUE HUA HONG,1TAI-VU TON,1STEPHANIE A. LAHOUSSE,1AND ROBERT C. SILLS1
1National Institutes of Environmental Health Sciences, Research Triangle Park, NC
Tumor response in the B6C3F1 mouse, F344 rat, and other animal models following exposure to various compounds provides evidence that peo-
ple exposed to these or similar compounds may be at risk for developing cancer. Although tumors in rodents and humans are often morphologically
similar, underlying mechanisms of tumorigenesis are often unknown and may be different between the species. Therefore, the relevance of an animal
tumor response to human health would be better determined if the molecular pathogenesis were understood. The underlying molecular mechanisms
leading to carcinogenesis are complex and involve multiple genetic and epigenetic events and other factors. To address the molecular pathogenesis of
environmental carcinogens, the authors examine rodent tumors (e.g., lung, colon, mammary gland, skin, brain, mesothelioma) for alterations in can-
cer genes and epigenetic events that are associated with human cancer. National Toxicology Program (NTP) studies have identified several genetic
alterations in chemically induced rodent neoplasms that are important in human cancer. Identification of such alterations in rodent models of chem-
ical carcinogenesis caused by exposure to environmental contaminants, occupational chemicals, and other compounds lends further support that they
are of potential human health risk. These studies also emphasize the importance of molecular evaluation of chemically induced rodent tumors for
providing greater public health significance for NTP evaluated compounds.
animal models; cancer; carcinogenesis; genotoxins/non-genotoxins; toxicologic pathology; rodent pathology; risk
Each year the National Toxicology Program (NTP) receives
nominations of substances deemed of potential human health
concern from federal, public, and other sources. The NTP then
reviews and selects compounds to be evaluated for potential
toxicologic or carcinogenic effects. These compounds range
from herbal supplements to occupational chemicals to environ-
mental contaminants. The B6C3F1 mouse, F344 rat, and other
mouse and rat models have been used as models to assess
effects of these compounds through multiple routes of expo-
sure, including feed, gavage, inhalation, and topical adminis-
tration, and pathology data gained from subchronic (90-day)
and chronic (2-year) NTP studies is used for hazard identifica-
tion to assess the toxicologic and carcinogenic effects of these
compounds in rodents.
Using available tissues from NTP studies that exhibit a
tumorigenic response, our laboratory evaluates genetic and epi-
genetic alterations in major classes of oncogenes and tumor
suppressor genes in spontaneous and chemically induced
rodent neoplasms. The data presented here includes the
evaluation of several NTP studies performed between 2004 and
2008 for which molecular studies were undertaken. Under-
standing the underlying molecular pathogenesis of cancer in
these studies will help determine if the response in the rodent
is similar to or different from that of humans. In this way, a
major goal of these studies was to relate oncogenic events that
occur in the rodent as a result of chemical exposure to changes
present in the human disease and make conclusions about
potential human cancer risk. Another goal of these studies was
to distinguish chemical-specific tumor responses from sponta-
neous events and to determine if signature mutation patterns
that occur in rodents following chemical exposure are similar
to patterns relevant to cancer in humans.
1. COLON CANCER
Colon cancer is the third leading cause of cancer deaths in
the United States. The pathogenesis of colon cancer is a multi-
step process involving activation of oncogenes, loss of tumor
suppressor gene function, and dysfunction of DNA repair. Loss
of function of the APC tumor suppressor gene or b-catenin
(CTNNB1) gene mutation are early events in the development
of colon cancer in humans (Bienz and Clevers 2000; Kinzler
and Vogelstein 1996; Peifer 1997). In the WNT signaling path-
way, APC forms a large protein complex with b-catenin, axin,
and glycogen synthase kinase-3b (GSK-3b) as a result of WNT
Address correspondence to Mark J. Hoenerhoff, National Institutes of
Environmental Health Sciences—Cellular and Molecular Pathology Branch,
111 TW Alexander Dr., Durham, NC 27519, USA; e-mail: hoenerhm@
Molecular Mechanisms and Toxicologic Pathology
Toxicologic Pathology, 37: 835-848, 2009
Copyright # 2009 by The Author(s)
ISSN: 0192-6233 print / 1533-1601 online
activation and ultimately results in b-catenin phosphorylation
and proteosomal degradation. With loss of function of APC due
to mutation, accumulation of b-catenin occurs, followed by
activation of nuclear transcription factors and increases in lev-
els of target proteins such as MYC and cyclin D1. However,
mutation of the CTNNB1 gene without loss of the APC gene
also results in accumulation of b-catenin protein and increased
WNT signaling in a subset of colon cancers (Davies, Miller,
and Coleman 2005; Fearon and Vogelstein 1990; Fodde, Smits,
and Clevers 2001; Mirabelli-Primdahl et al. 1999). The genetic
alterations involved in familial adenomatous polyposis (FAP),
leading to colorectal cancer, have been well documented, and
in addition to alterations of the APC gene, there are mutations
in the KRAS oncogene, p53 tumor suppressor gene (TP53), and
TGFB growth regulatory genes (Bos et al. 1987; Fearon and
Vogelstein 1990; Johnstone, Chang, and Ernst 2002). In addi-
tion to known genetic causes, environmental factors may con-
tribute to the estimated 130,000 new cases of colon cancer per
year in the United States (Sills et al. 2004).
o-Nitrotoluene-Induced Large Intestinal Cancer
o-nitrotoluene is used to synthesize agricultural and rubber
products; azo and sulfur dyes; and dyes for cotton, wool, silk,
leather, and paper (Dunnick et al. 2003; Huff et al. 1985).
Low-level contamination of rivers and drinking water has been
determined through environmental surveys (Sills et al. 2004).
Given the fact that o-nitrotoluene is a known rodent carcinogen
and that low levels of this contaminant exist in the environment,
the chemical clearly poses a potential human health risk.
The National Toxicology 2-year carcinogenicity bioassay
determined that exposure of B6C3F1 mice to o-nitrotoluene
resulted in an increased incidence of cecal carcinomas (NTP
2002). This exposure model provided the first cecal tumor
response in mice and an opportunity to evaluate the morphology
that have relevance to the human disease (Sills et al. 2004). The
induced mouse carcinomas were morphologically and immuno-
histochemically similar to human colonic adenocarcinoma
(Goldstein et al. 2000), characterized by clusters and tubules
of poorly differentiated, CK7 positive, CK20 negative, tall
columnar epithelial cells that invaded into and through the mus-
cle wall. Furthermore, accumulation of b-catenin protein was
detected in 80% of the mouse cecal carcinomas, and 73% of the
immunohistochemistry. There was no significant change in the
expression of APC protein in the cecal carcinomas.
Mutational analysis revealed multiple mutations of the
Kras, Tp53, and b-catenin (Catnb) genes in cecal carcinomas
from B6C3F1 mice exposed to o-nitrotoluene. Exon 2
TABLE 1.—Summary of genetic mutations in National Toxicology Program (NTP) molecular pathology studies.
Organ ChemicalSpecies/strain Tumor type
mutation Ancillary testing
o-nitrotoluene mouse/B6C3F1Cecal carcinoma K-Ras
IHC: CK7þ, CK20-, B-cateninþ, Cyclin
Dþ, P53þ (Sills et al. 2004)
Lung Cumenemouse/B6C3F1AB adenoma/carcinoma Microarray: "ERK-MAP kinase, "HDACs,
"genes associated with invasion, metastasis,
#tumor suppressor genes, genes associated
with inhibition of migration, invasion, and
(Hong et al. 2008)
(Ton et al. 2007)Lung 1,3-butadiene chloroprenemouse/B6C3F1AB adenoma/carcinoma K-Ras
AB adenoma/carcinoma(Hong, Dunnick, et al. 2007)
HCA, HCC, HB
(Hong, Houle, et al. 2007)
IHC: Cyclin Dþ, nuclear B-catenin, loss of
Western blot: "cyclin D1, B-catenin deletion
(Devereux et al. 1999, Hayashi et al. 2003,
Anna et al. 2003)
(Hong, Houle, et al. 2007) Harderian
Ethylene oxidemouse/B6C3F1Papillary cystadenoma/
Glioma and neuroblastoma
(Hong, Houle, et al. 2007)
(Ton et al. 2007)
Squamous cell carcinoma
IHC: p53þ, PCNAþ (Lambertini et al. 2005)
Microarray: dysregulation of Igf, p38
MAPK, Wnt/B-catenin, and integrin
signaling pathways (Kim et al. 2006)
836 HOENERHOFF ET AL.TOXICOLOGIC PATHOLOGY
(corresponding with exon 3 in humans) Catnb mutations were
present in 100% of carcinomas, and mutations in Tp53 and
Kras occurred in a majority of tumors (Sills et al. 2004). These
data showed that genetic alterations in the mouse large intest-
inal carcinomas resulted in activation of signal transduction
(Kras and Catnb) and disruption of cell-cycle control (Tp53,
cyclin D1), hallmarks of human colon cancer. Considering that
100% of cecal carcinomas had mutations in the Catnb gene, it
is likely that dysregulated expression of b-catenin protein plays
a role in the pathogenesis of these mouse cecal carcinomas, as
is the case in human colon cancer.
These data correlate with the hypothesis that large intestinal
epithelium in the mouse, similar to the case in humans, must
acquire multiple mutations for transformation, ultimately
culminating in the full malignant phenotype of the adenocarci-
noma. The activation of the Kras oncogene provides growth-
promoting signals, the loss of Tp53 results in unregulated
growth and DNA repair defects, and activation of both Catnb
Weinberg 2000; McCormick 1999). The interaction between
these various mutations and their impact on signaling pathways
ultimately culminates in the full malignant phenotype of cecal
2. LUNG CANCER
Lung cancer is the most commonly diagnosed cancer in the
world and the leading cause of cancer death worldwide (Hong
et al. 2008). Lung cancer can be divided into non-small-cell
lung cancer (80-85%) and small-cell lung cancer (15-20%)
based upon histopathologic features (Herbst, Heymach, and
Lippman 2008; Wakamatsu et al. 2007). Non-small-cell lung
cancer can be further divided into adenocarcinoma, squamous
cell carcinoma, and large-cell carcinoma, of which adenocarci-
noma is the most prevalent (Husain et al. 2005; Wakamatsu
et al. 2007). Most lung cancers in humans are non-small-cell
lung cancers resulting from genetic and epigenetic damage
from chronic exposure to tobacco smoke carcinogens (Hecht
1999; Herbst, Heymach, and Lippman 2008), but the frequency
of adenocarcinoma is on the rise in nonsmokers (Husain et al.
2005). The pathophysiology of pulmonary tumors in general is
complex, and relatively little is known about the exact genetic
mechanisms underlying the earliest stages of this multistep pro-
cess. Mouse models have been utilized for studying carcino-
genesis of human lung cancers and are both histologically
similar and share many of the major genetic alterations
detected in human pulmonary adenocarcinoma (Wakamatsu
et al. 2007), including activation of the KRAS proto-oncogene
(Meuwissen and Berns 2005; Nikitin et al. 2004) and inactiva-
tion of the TP53 tumor suppressor gene (Horio et al. 1996;
Jackson et al.2005;Takahashi et al.1989). Krasmutation is the
most common molecular alteration identified in mouse lung
adenomas and carcinomas (Ton et al. 2007) and occurs in
30% to 50% of human lung adenocarcinomas. The cumene
mouse model of lung carcinogenesis recapitulates many of the
molecular alterations found in the human disease.
Cumene-Induced Lung Tumors in B6C3F1 Mice
Cumene, or isopropylbenzene, is a component of crude oil
used primarily in the production of acetone and phenol (Hong
et al. 2008). Since the annual production of cumene is increas-
ing in the United States, and there is significant occupational
exposure by inhalation, it was nominated for study by the NTP.
The 2 year cumene carcinogenicity bioassay demonstrated a
significant increase in the incidence of alveolar/bronchiolar
adenomas and carcinomas in B6C3F1 mice (Hong et al.
2008; NTP 2007). Since certain chemicals often induce spe-
cific patterns of activation of Ras oncogenes or inactivation
of Tp53 tumor suppressor gene function compared to sponta-
neous neoplasms in mice (Sills et al. 2004; Sills, Hong, et al.
1999), and these genetic mutations are considered important
in the pathogenesis of human lung cancer, we evaluated spon-
taneous and cumene-induced lung neoplasms for mutations in
Kras and Tp53 genes, as well as chemical-specific mutations
that could function as biomarkers of chemical exposure with
potential human health importance.
Cumene-induced tumors had a significantly higher inci-
dence of Kras mutations (87% vs. 28%) and Tp53 mutations
(52% vs. 0%) compared to spontaneous lung tumors (Hong
et al. 2008). While alterations in TP53 protein were not
observed in normal lung or spontaneous lung tumors, increased
TP53 protein expression was detected by immunohistochemis-
try in 56% of cumene-induced neoplasms. Additionally, there
was loss of heterozygosity (LOH) on chromosome 4 near the
p16 gene and on chromosome 6 near the Kras gene in a subset
of cumene-induced lung carcinomas, while there was no LOH
observed in spontaneous carcinomas or normal lung tissue
(Hong et al. 2008).
To distinguish gene expression patterns based on the
presence or absence of Kras and Tp53 mutation, spontaneous
and cumene-induced tumors were analyzed by microarray
(Wakamatsu et al. 2008). Principal component analysis was
able to segregate pulmonary carcinomas induced by cumene
into groups with and without Kras mutations. Following this,
genes associated with the ERK-MAPK signaling pathway were
shown to be significantly upregulated in tumors with Kras
mutations, as well as a number of genes associated with tumor
malignancy (invasion and metastasis) such as Krt8, Krt18,
Lasp1, Sdc1, Ccnd1, and Mmp14, suggesting that cumene-
induced carcinomas with Kras mutations have greater malig-
nant potential than those without mutations (Wakamatsu
et al. 2008). SAFE (significance analysis of function and
expression) analysis further showed that carcinomas with Kras
mutations had changes in expression of histone deacetylases
(HDACs), suggesting histone modification as an epigenetic
mechanism of carcinogenesis in cumene-induced lung carcino-
mas (Wakamatsu et al. 2008).
Our gene expression analysis suggested the formation of
alveolar/bronchiolar carcinomas in cumene-exposed mice typi-
cally involves mutation and activation of Kras, with activation
of the MAPK pathway and dysregulation of a number of genes
associated with tumor malignancy. In cumene-induced tumors
Vol. 37, No. 7, 2009MECHANISMS OF CANCER IN NTP RODENT BIOASSAYS 837
with and without Kras mutation, there was downregulation of a
number of tumor suppressor genes (Ptprd, Igsf4a, Fh11, Pdzd2,
Cdkn2d, Cdh5, Lox11, and Akap12) and genes associated with
inhibition of invasion (Reck, Gsn, Lims2, Cav1, and Gpx3). In
cumene-induced tumors with Kras mutations, there was
decreased expression of additional genes associated with inhi-
bition of cell motility and cell proliferation (Igfbp4, Sod3, Rb1,
Cebpd, Vwf, and Dlc1) and genes associated with patient sur-
vival (Cyr61 and Enpp2). Alternatively, there was increased
expression of genes known to increase invasion and metastasis
(Krt18, Krt8, Lasp1, Mif, Mmp14, and Tacstd1), inhibit apop-
tosis (Areg, Cks1b), increase angiogenesis (Slc2a1, Gnb211,
and Ptges), and increase metastatic potential (Sdc1 and Ccnd1)
(Wakamatsu et al. 2008). Additionally, cluster analysis of
genes generally associated with HDAC regulation revealed a
stronger association with tumors with Kras mutation.
These results suggest that in cumene-induced pulmonary
tumors in mice, DNA damage and genomic instability leading
to Kras and Tp53 dysregulation leads to upregulation of path-
ways associated with the development of lung cancer in
cumene-exposed mice and that tumors resulting from mutations
in Kras possess a gene expression associated with a greater
degree of malignancy. Additionally, the molecular alterations
in cumene-induced lung tumors appear to affect similar path-
ways as those in the human disease, suggesting that the tumor
response in mice may be relevant to human lung cancer.
AZT-Induced Lung Tumors in CD-1 Mice
3-azido-3-deoxythymidine (AZT) was the first drug
approved by the U.S. Food and Drug Administration (FDA) for
treatment of HIV-1 in adults and children (Bhana et al. 2002;
Vivet-Boudou et al. 2006). AZT also reduces vertical transmis-
sion of HIV-1 during pregnancy by 70% (Connor et al. 1994).
However, there are concerns about its carcinogenic potential,
given that it induces gene mutations and chromosomal damage
both in vitro and in laboratory animals via direct or in utero
exposure (Olivero et al. 1997; Phillips et al. 1991).
(CD-1) mouse offspring had a significantly increased incidence
of alveolar/bronchiolar adenomas and carcinomas (NTP
were evaluated for Kras and Tp53 point mutations by DNA
sequencing of formalin-fixed, paraffin-embedded tumors. The
results of mutational analysis revealed that a majority of the
AZT-induced tumors contained mutations in Kras (66%) and
Tp53 (84%) (Hong, Dunnick, et al. 2007). The primary Kras
mutationwasa G!Ttransversionatcodon12,andthe predomi-
nant Tp53 mutations were A!T transversions in exon 8, codon
285 and T!A transversions in exon 6, codon 198. Although the
tically significantly increased above controls, a majority of these
mice (Hong, Dunnick, et al. 2007).
Our laboratory determined that AZT-induced pulmonary
tumors in both male and female CD-1 mice resulted from mole-
cular alterations in the same pathways as human lung cancer
and that genotoxic damage from AZT or its metabolites may
contribute to the development of lung tumors in these mice
(Hong, Dunnick, et al. 2007; Koujitani et al. 2008). This sug-
gests that the response in the mouse may be of relevance to the
mechanism of carcinogenicity in humans. In addition, these
data are in agreement with other transplacental carcinogenicity
studies (Olivero et al. 2001), in vitro models (Meng et al. 2002,
2000) and rodent (Diwan et al. 1999; Poirier et al. 2003; Von
Tungeln et al. 2002) and human (Olivero et al. 1999) fetuses
exposed to AZT. Taken together, these data suggest that human
infants exposed to AZT in utero may have an increased risk of
lung cancer later in life.
Ethylene Oxide–Induced Lung and Other Tumors in
Ethylene oxide (EO) is commonly used as a sterilization
agent in the health care industry, and it is also used to produce
several chemicals. As a result of occupational exposure in the
health care industry, it is estimated that 75,000 workers are at
risk of exposure (IARC [International Agency for Research
on Cancer] 1994; NTP 2004b; Recio et al. 2004). It has carci-
nogenic activity in rodents, where it induces tumors in the lung,
cells (IARC 1994; NTP 1987; Nygren et al. 1994). Chronic
human exposures have been suggested to be associated with leu-
kemias, lymphomas, breast cancer, and stomach cancer (Norman
et al. 1995; Shore, Gardner, and Pannett 1993; Stayner et al.
1993; Steenland et al. 1991; Teta, Benson, and Vitale 1993).
To evaluate the potential human risk from EO, our labora-
tory examined EO-induced tumors in the lung, harderian gland,
and uterus from exposed mice for Kras mutations. While this
mutation was found in 25% (27/108) of spontaneous lung
tumors, 100% (23/23) of EO-induced lung tumors had Kras
mutations (Hong, Houle, et al. 2007). The most common muta-
tions were codon 12 G!T transversions, which were infre-
quent in spontaneous tumors. Kras mutations were also very
common in EO-induced harderian gland tumors (18/21, 86%)
and infrequent in spontaneous tumors (2/27, 7%). In EO-
induced tumors, codon 13 G!C and codon 12 G!T transver-
sions were common, but spontaneous tumors did not possess
these mutations. Likewise, in uterine tumors, Kras mutations
occurred in a majority (5/6, 83%) of EO-induced uterine carci-
nomas, and all were codon 13 C!T transitions.
an important step in the pathogenesis of EO-induced lung, hard-
erian gland, and uterine tumors in the B6C3F1 mouse (Hong,
ther evaluation of its effects in humans is warranted.
838 HOENERHOFF ET AL.TOXICOLOGIC PATHOLOGY
1,3-Butadiene- or Chloroprene-Induced Lung and Brain
Tumors from B6C3F1 Mice
1,3-butadiene and chloroprene are used in the production of
synthetic rubber (Himmelstein et al. 1997; NTP 1993, 1998).
Additionally, 1,3-butadiene is a combustion product of fossil
fuel and wood and is present in automobileexhaust and tobacco
smoke. Both chemicals are multiorgan carcinogens in rodents,
most often causing lung tumors in female mice (Huff et al.
1985; Melnick et al. 1999). Other tumors induced by these che-
micals include lymphoma, hemangiosarcoma, and harderian
gland tumors. Epidemiological studies in humans have corre-
lated occupational exposure to 1,3-butadiene with increased
leukemia and lymphoma incidence (Macaluso et al. 1996;
Santos-Burgoa et al. 1992). Based upon these and other in vitro
and in vivo findings, the NTP Report on Carcinogens (ROC)
has listed 1,3-butadiene as a ‘‘known human carcinogen’’ and
chloropene as ‘‘reasonably anticipated to be a human carcino-
gen’’ (NTP 2000).
Mutational analysis indicated that a large proportion of lung
tumors induced by these chemicals harbored Kras mutations
(Ton et al. 2007). A majority (34/51, 67%) of lung tumors
induced by 1,3-butadiene had codon 13 G!C transversions.
Likewise, a large proportion (19/25, 76%) of lung tumors aris-
ing from chloroprene exposure had Kras mutations, except that
mutation profile was different, with half of the tumors harbor-
ing codon 61 A!T mutations. These mutations lead to the for-
mation of guanine and adenine DNA adducts in the lung and
may play critical roles in the development of lung tumors
induced by these chemicals. In addition to Kras mutations,
there was a high frequency of loss of heterozygosity (LOH)
in these tumors in the region of Kras on chromosome 6, sug-
gesting loss of the wildtype allele. The findings of Kras muta-
tion and wildtype allele loss in these mouse lung tumors are
similar to molecular alterations in some human lung adenocar-
cinomas and suggest that wildtype Kras may act as a tumor
suppressor gene (Zhang et al. 2001). Further inhalation studies
of 1,3-butadiene and chloroprene may provide a model system
for understanding certain types of lung cancer in humans.
Along with the increased incidence of lung tumors, harder-
ian gland tumors, hemangiosarcomas, and lymphomas, expo-
sure to 1,3-butadiene surprisingly resulted in an increased
incidence of brain tumors in B6C3F1 mice (Kim et al. 2005).
While the central nervous system is a rare target for carcino-
genesis in NTP bioassays (Ton et al. 2007), there was an
increase in glioma and neuroblastoma formation with loca-
tional and morphologic similarities to their human counterparts
(Kleihues et al. 2002). Many of the molecular alterations in
brain tumors resulting from 1,3-butadiene exposure in mice are
similar to that seen in human brain tumors. First, alterations in
the Tp53 tumor suppressor gene were common. TP53 mutation
is common in human glial tumors, dysfunction of which has
been suggested to be an early event in glial tumorigenesis in
humans (Holland 2001). There were mis-sense mutations in
half (3/6) of malignant gliomas and in both (2/2) neuroblasto-
mas examined. These tumors were always associated with loss
of heterozygosity of the Tp53 gene. Most of the Tp53 mutations
found in this study were G!A transitions, similar to those seen
in human glioblastoma, in which up to 67% of tumors have
Tp53 mutations, with the majority being G!A transitions
(Fulci et al. 2000; Watanabe et al. 1996). Loss of the P16
(CDKN2A) tumor suppressor gene has been observed in human
astrocyte cancer cell lines in vitro (Bachoo et al. 2002) and
similarly, LOH was noted at the Ink4a/Arf tumor suppressor
locus in all mouse gliomas and neuroblastomas, indicative of
loss of the Cdkn2a tumor suppressor gene. These data show
that the molecular changes induced by exposure to 1,3-
butadiene are similar to those present in the human disease and
suggest that environmental exposure to this chemical might
pose a significant cancer risk. This is the first report to our
knowledge in which multiple mutations of tumor-associated
genes observed in human brain tumors have been detected in
mouse brain tumors following exposure to an environmental
3. SKIN CANCER
Squamous cell carcinoma (SCC) is the second most com-
mon skin cancer in humans, accounting for 20% of all malig-
nancies of the skin (McGuire, Ge, and Dyson 2009; Wade
and Ackerman 1978). Ultraviolet light plays a significant role
in the development of SCC, and both UVA and UVB may con-
tribute to skin cancer; both UVA and UVB cause DNA dam-
age, but UVB also causes injury to Langerhans cells,
resulting in compromised local immune surveillance (de Gruijl
2000; McGuire, Ge, and Dyson 2009). Other common causes
of SCC include thermal injury, human papillomavirus, chronic
irradiation dermatitis, and chemical carcinogenesis (McGuire,
Ge, and Dyson 2009).
Squamous Cell Carcinomas in HRA/Skh Mice Induced by
8-Methoxypsoralen (8-MOP) and UVA Radiation
Treatment with 8-methoxypsoralen (8-MOP) and ultraviolet
radiation (primarily UVA), called PUVA therapy, has been
used for decades to treat various skin diseases such as psoriasis,
vitiligo, and cutaneous T-cell lymphoma. The mechanism of
action is through photosensitization by psoralen, which cova-
lently binds pyrimidine bases through a photocycloaddition
reaction following UVA exposure (Gasparro, Felli, and
Schmitt 1997). However, epidemiological evidence has shown
a significant increase in skin tumors, predominantly SCC, in
chronically treated individuals. A consistent mutation pattern
of TP53 has been shown in vivo and in vitro as a result of the
direct action of UV light on DNA (Inga et al. 1998; Monti et al.
2000; Nataraj et al. 1997; Santamaria et al. 2002), rather than
photoactivation of the psoraslen treatment, showing a direct
involvement of the TP53 gene in human skin tumorigenesis.
The Hras proto-oncogene is also known to play a role in
chemically induced skin carcinogenesis in rodents (Mangues
and Pellicer 1992) and is a known mutational target in
PUVA induced SCC in humans (de Gruijl, van Kranen, and
Mullenders 2001; Sills, Boorman, et al. 1999).
Vol. 37, No. 7, 2009 MECHANISMS OF CANCER IN NTP RODENT BIOASSAYS839
The NTP performed a study in mice exposed to PUVA ther-
apy to test its carcinogenic potential (Dunnick et al. 1991). The
results showed that PUVA therapy caused a significant
increase in SCC of the skin in the hairless HRA/Skh mouse.
Our laboratory then examined the Tp53 and Hras mutational
pattern as well as Tp53 and PCNA protein expression in hyper-
plastic and neoplastic squamous cell lesions from the NTP
study (Lambertini et al. 2005). By immunohistochemistry,
Tp53 and PCNA protein was detected in 3/16 (19%) of hyper-
plastic lesions and 14/17 (82%) of SCCs in animals that were
treated with both 8-MOP and UVA (Lambertini et al. 2005).
In UVA and 8-MOP treated animals with SCC, 15/17 (88%)
had mutation of Tp53, and 93% of those animals had mutation
in exon 6. However, Tp53 mutations in SCCs from human
patients treated with PUVA predominantly occur in exons 5,
7, and 8. Additionally, therewas no evidence of Hrasmutations
in either hyperplastic skin lesions or SCCs. Overexpression of
Tp53 and PCNA protein was not observed in normal mouse
The study described above showed that photoactivated
8-MOP induced an increased incidence of SCCs with a high
frequency of Tp53 mutations in HRA/Skh mice that were dosed
orally and given similar UV intensity as would occur in human
patients (Dunnick et al. 1991; Lambertini et al. 2005; Nataraj
et al. 1997). The mutagenic effect of PUVA on the Tp53 tumor
suppressor gene may lead to a conformational modification and
inactivation of the Tp53 protein, which is considered a critical
step in PUVA-induced skin carcinogenesis. Although the Tp53
mutational frequency and patterns were different from those
reported in PUVA-type tumors, the carcinogenic risk of PUVA
treatment should not be underestimated, and preventative
measures should be taken when this clinical approach is used
(Lambertini et al. 2005).
4. MALIGNANT MESOTHELIOMA
In humans, mesothelioma is a malignant proliferation of the
lining of body cavities, most commonly the pleura, but also
occurs in the peritoneum and pericardium as well as the tunica
vaginalis (Musti et al. 2006; Spugnini et al. 2006). Human
malignant mesothelioma is most commonly caused as a result
of exposure to asbestos (Carbone and Bedrossian 2006; Suzuki
and Yuen 2002). However, whether asbestos induces mesothe-
lioma by direct interaction with pleural cells or indirectly
through the generation of toxins and reactive oxygen species
that may result in genotoxicity or dysregulation of other cellu-
lar pathways is not clear.
The ability for asbestos to stimulate mesothelial prolifera-
tion through induction of AP-1 and activation of NF-kB
through TNFa released by macrophages in response to inflam-
mation has been established (Carbone and Bedrossian 2006).
However, development of human malignant mesothelioma is
mediated by several additional genetic defects that result in
loss or downregulation of tumor suppressors or overexpression
of oncogenes. Loss of cyclin-dependent kinase function
(CDKN2A, CDKN2B), leading to Tp53 and pRB inhibition,
is well characterized in human malignant mesothelioma, as is
overexpression of genes associated with cellular growth and
survival (IGF, IGFR, EGFR, FOS, JUN), anti-apoptotic path-
ways (BCL2), angiogenesis (VEGF, COX2), and loss of
tumor suppressor genes either through mutation (NF2, WT1)
or hypermethylation (APC, CDNK2A, CDNK2B, RASSF1A)
(Christensen et al. 2008; Kumar and Kratzke 2005; Spugnini
et al. 2006; Whitson and Kratzke 2006).
Acid-Induced Peritoneal Mesothelioma in F344 Rats
This study was performed to identify and characterize major
carcinogenic pathways involved in rat peritoneal mesothelioma
(RPM) formation following treatment with o-nitrotoluene
(o-NT) or bromochloracetic acid (BCA) in F344 rats (Kim
et al. 2006). In the F344 rat, spontaneous mesothelioma occurs
at an incidence of 2.7% to 3.6% and primarily affects the peri-
toneal cavity and, more rarely, the thoracic cavity (Haseman,
Arnold, and Eustis 1990; Kim et al. 2006). Many chemicals
in NTP studies have been shown to increase the incidence of
mesothelioma in F344 rats, including o-NT and BCA (NTP
2002). Tumors are induced by o-NT in multiple sites, and our
laboratory has previously shown that o-NT-induced cecal car-
cinomas have alterations in the WNT/Beta-catenin signaling
pathway, KRAS/MAP kinase pathway, Tp53 pathway, and
cyclin D1 (Sills et al. 2004).
This study was conducted to determine the major carcino-
genic pathways at play in the development of RPMs due to
o-NT or BCA exposure in F344 rats. More than 20,000 genes
were evaluated in chemically induced rat peritoneal mesothe-
liomas using oligo arrays and comparing the data to a nontrans-
dysregulation of 169 cancer-related genes, involving numerous
biological processes such as cell cycle progression, growth and
proliferation, apoptosis, invasion, and metastasis. Importantly,
there are many pathways important in the development of
mesothelioma in humans that were subsequently identified
in chemically induced rat mesotheliomas, including insulin-
like growth factor 1 (Igf1), p38 MAPK (Mapk14), WNT/
Beta-catenin, and integrin signaling pathways (Kim et al.
2006). This demonstrates that RPMs induced by o-NT and BCA
are similar to the human disease at the cellular and molecular
level, providing an opportunity to identify genetic pathways that
may be of importance in the study of the human disease.
5. LIVER CANCER
Hepatocellular carcinoma (HCC) accounts for more than
90% of primary hepatic neoplasia (Kim, Sills, and Houle
2005). The pathogenesis of this disease in humans is multifac-
torial, associated with various infectious agents, carcinogens,
and environmental and lifestyle factors (Coleman 2003). In
B6C3F1 mice, hepatocellular adenoma (HCA) is the most
common spontaneous liver neoplasm second to HCC and
occurs more commonly in males than in females (Kim, Sills,
and Houle 2005). Hepatoblastoma (HB) is a malignant
840HOENERHOFF ET AL.TOXICOLOGIC PATHOLOGY
embryonal tumor affecting predominantly children under 3
years of age (Koch et al. 1999). It not only is the most common
malignant hepatic tumor in children, accounting for 1.5 cases
per 1 million, occurring most often sporadically, but also is
associated with familial syndromes such as familial adenoma-
tous polyposis coli (FAP) or Beckwith-Wiedemann syndrome
(Ishak and Glunz 1967; Kim, Sills, and Houle 2005; Steenman,
Westerveld, and Mannens 2000). Hepatoblastomas in mice
rarely occur spontaneously, while higher incidences may occur
in chemically induced models and may occur within a preexist-
ing HCC or HCA (Turusov et al. 2002). Various genetic altera-
tions have been reported in the development of liver tumors in
humans, including alterations in genes involved in growth and
proliferation, oncogenes (Coleman 2003), DNA damage
response and cell cycle control genes (Hainaut et al. 1998),
genes associated with cell-cell interaction and signal transduc-
tion (Nhieu et al. 1999; Yamamoto et al. 2003), as well as epi-
genetic mechanisms (Cherian, Jayasurya, and Bay 2003; Lim
2002; Wong et al. 2003). Many of the same genes involved
in human hepatocarcinogenesis are implicated in the develop-
ment of altered foci, HCA and carcinoma, and HB in mice,
emphasizing the importance of the mouse in the study of the
human disease (Kim, Sills, and Houle 2005). For example, pre-
neoplastic proliferative liver lesions in humans have increased
expression of several growth factors and receptors (IGFII,
HGF, TGFA) (Coleman 2003; Kiss et al. 1997; Lund et al.
2004) that have been shown to cause liver tumors in transgenic
mouse models (Fausto 1999; Kim, Sills, and Houle 2005).
Furthermore, liver tumors may be associated with mutations
in oncogenes such as Hras in spontaneous or chemically
induced mouse models (Kim, Sills, and Houle 2005), and
increased expression of HRAS, NRAS, and KRAS have been
associated with liver tumors and preneoplastic lesions in
humans, with associated upregulation of downstream MAP
kinase pathway (Coleman 2003). Alterations in the WNT sig-
naling pathway are common in chemically induced liver
tumors, including mutations in b-catenin (Catnb), resulting in
translocation of b-catenin to the nucleus and cyclin D expres-
sion (Anna et al. 2003; Kim, Sills, and Houle 2005). Mutations
in the b-catenin gene are frequent and early in the pathogenesis
of chemically induced hepatic tumors in mice and may be
chemical-specific in nature (Devereux et al. 1999). In the NTP
2-year bioassay of methyleugenol-induced and oxazepam-
induced liver tumors in B6C3F1 mice, there was a 69% and
41% mutation incidence in b-catenin, respectively (Anna
et al. 2003; von Schweinitz et al. 1996). Similarly, following
exposure to diethanolamine for 2 years, B6C3F1 mice devel-
oped HCC associated with genetic alterations in the Catnb
gene; 32% (11/34) adenomas and carcinomas had mutations
in exon 2 of the b-catenin gene (Hayashi et al. 2003). Similar
mutations in exon 3 of the human b-catenin gene have been fre-
quently reported in human HCA (Nhieu et al. 1999; Yamamoto
et al. 2003). Like HCA, hepatoblastomas in mice and humans
are also associated with high incidences of b-catenin gene
mutation (Anna et al. 2000; Jeng et al. 2000; Koch et al.
1999), including downstream activation of WNT signaling
(Anna et al. 2003; Takayasu et al. 2001). In B6C3F1 mice,
100% (5/5) of hepatoblastomas and 34% (12/35) of HCA had
elevated expression of cyclin D1, including 67% (10/15) HCA
with b-catenin gene mutation (Anna et al. 2003). Other media-
tors associated with or involved in the regulation of b-catenin
(EGFR, MET, CDH1) are altered in both human and mouse
hepatoblastoma (Anna et al. 2003; von Schweinitz et al.
1996), illustrating an overreaching role of WNT signaling on
6. BREAST CANCER
According to the American Cancer Society, breast cancer is
the most common cancer in women in the United States and is
the second leading cause of cancer mortality (Jemal et al.
2008). Several genetic and epigenetic alterations have been
implicated in the genesis of breast cancer, and in general breast
cancer is divided into hereditary and sporadic forms. Heredi-
tary forms account for approximately 5% to 10% of breast
cancers and are caused predominantly by mutation in the
high-penetrance breast cancer susceptibility genes BRCA1 and
BRCA2 (Tan, Marchio, and Reis-Filho 2008). Sporadic or non-
hereditary cases of breast cancer are associated with a variety
of genetic and epigenetic abnormalities that result in dysregu-
lation of growth pathways (IGF, EGFR, ESR1), cell cycle reg-
ulators (CDKN1A, CDKN1B, CDKN2A, CCND1), oncogenes
(ERBB2, MYC), tumor suppressor genes (TP53, RB, ATM,
CDH1), and chromatin modifiers (BMI1) (Datta et al. 2007;
Kenemans, Verstraeten, and Verheijen 2004; Lerebours and
Lidereau 2002). While RAS is mutated in less than 10% of
breast cancers (Lerebours and Lidereau 2002), the RAS/RAF/
MEK/ERK growth signaling pathway is reported to be fre-
quently activated in breast cancer (McCubrey et al. 2007).
P53 and H-ras Mutations in Benzene- and Ethylene
Oxide–Induced Mammary Carcinoma in B6C3F1 Mice
Benzene and ethylene oxide have been shown to cause can-
cer at multiple sites in rodents and have been classified by the
NTP as human carcinogens. Both chemicals have been shown
to cause increased incidence of mammary carcinomas in mouse
carcinogenicity studies (IARC 1994; Maltoni and Scarnato
1979; NTP 2004a; Snellings, Weil, and Maronpotet 1984).
TP53 and RAS are the most frequently mutated genes in human
cancers, and based on a high frequency of TP53 mutation and
RAS signaling in human breast cancers, this study was per-
formed to determine the role of Tp53 and Hras in spontaneous,
benzene-, and ethylene oxide–induced mouse mammary carci-
nomas in B6C3F1 mice.
TP53 protein expression was detected in a significant pro-
portion of spontaneous (42%), benzene-induced (6/14, 43%),
and ethylene oxide–induced (8/12, 67%) mammary carcinomas
by immunohistochemistry (Houle et al. 2006). The amount of
Tp53 protein detected by semiquantitative analysis was
fivefold to sixfold higher in chemically induced carcinomas
compared to spontaneous tumors. DNA mutation analysis
detected Tp53 mutation in a significant number of spontaneous
Vol. 37, No. 7, 2009MECHANISMS OF CANCER IN NTP RODENT BIOASSAYS 841
(7/12, 58%), benzene-induced (8/14, 57%), and ethylene
oxide–induced carcinomas (8/12, 67%). Twenty-six percent
(5/19) of spontaneous, 50% (7/14) of benzene-, and 33%
(4/12) of ethylene oxide–induced carcinomas had mutations
whenHrasmutations were present.Concurrent Tp53genemuta-
of ethylene oxide–induced, and 40% (2/5) of spontaneous carci-
nomas (Houle et al. 2006).
The mutation pattern between chemically induced and spon-
taneous tumors was significantly different. In chemically
induced tumors, Hras mutations most commonly involved the
second base of codon 61, and in 10/11 tumors resulted in an
amino acid change from glutamine to leucine or arginine.
Mutations in spontaneous tumors, however, involved the first
base and most often resulted in amino acid changes of gluta-
mine to lysine (Houle et al. 2006).
Our results show that although mutations of the Tp53 and
Hras genes are relatively common in spontaneous, as well as
in chemically induced, mouse mammary carcinomas, the inci-
dence of concurrent Tp53 and Hras mutation is increased in
chemically induced tumors (Houle et al. 2006). Furthermore,
the pattern of mutations in Tp53 and HRas differed between
chemically induced and spontaneous mammary carcinomas,
suggesting that different mechanisms are involved between
these tumors in B6C3F1 mice.
IMMUNOHISTOCHEMICAL DETECTION OF MOLECULAR ALTERATIONS
Immunohistochemistry may be used to detect the effects of
expression, loss, or abnormal localization may provide evidence
Abnormal Localization: As stated previously, the majority of
hepatoblastomas in mice have mutations in the b-catenin gene
(Anna et al. 2000; Kim, Sills, and Houle 2005). This mutation
affects the binding region of GSK3B, resulting in prevention of
phosphorylation of b-catenin and resultant accumulation within
the cytoplasm. Cytoplasmic b-catenin that is not targeted for
degradation by the proteasome may translocate to the nucleus,
where it may associate with LEF1/TCF transcription factors and
drive transcription of target genes such as Ccnd1 and Myc.
Therefore, evidence of this mutation may be seen with immuno-
histochemistry as both loss of membrane associated b-catenin
protein expression as well as abnormal localization to the
nucleus (Hayashietal.2003).Since nuclearlocalizationmaynot
be present in all tumors with Catnb mutation in mice (Devereux
et al. 1999), mutation analysis including PCR (polymerase chain
reaction) and sequencing should be used to follow up on the
identification of the specific mutation.
Overexpression: Gene mutation is often associated with over-
expression of a protein product. Several proto-oncogenes are
converted to oncogenes through mutationand induce
uncontrolled proliferation through various growth pathways.
The Ras proto-oncogenes undergo mutation in several different
types of cancer, resulting in downstream activation of numer-
ous kinases and anti-apoptosis mediators controlling the prolif-
eration of cells. Using immunohistochemistry, the effects of
Ras mutation may be detected, such as overexpression of
downstream mediators such as MAP kinases and transcription
factors that lead to promotion of the cell cycle and cell prolif-
eration (Sills, Boorman, et al. 1999). Western blot analysis
should be used to quantify the degree of overexpression,
and PCR and gene sequencing targeting the gene hot spots of
H-, N-, or Kras may be performed to identify the specific
Loss of Expression: Loss of expression of a protein product in
tumors is often associated with inactivation or deletion muta-
tions in tumor suppressor genes or genes regulating apoptosis.
The loss of tumor suppressor function results in loss of cell
cycle control and unregulated proliferation. Important tumor
suppressors often lost in tumorigenesis are Tp53 and retino-
blastoma (Rb). TP53 is the most commonly mutated gene in
human cancer. As the ‘‘guardian of the genome,’’ TP53 is
responsible for cell cycle control and arrest at the G1 check-
point to allow time for DNA repair. Mutation of Tp53 often
results in a loss of function of one or both alleles, resulting in
a dysregulation of this checkpoint arrest, and continuation
through the cell cycle without DNA repair. This allows
further mutations to accumulate and results in oncogenic trans-
formation as a result of defective DNA repair. The expression
of wildtype TP53 protein in tissues is transient, so it is unde-
tectable by immunohistochemical methods; however, when
mutated, a defective, nonfunctional TP53 protein product,
which has a long half-life, may accumulate within the nucleus
(Gerbes and Caselmann 1993). Therefore, loss of function of
the wildtype TP53 protein and overexpression of the mutated
form is indicative of genetic mutation. Mutation of other tumor
suppressor genes, such as Cdkn2a or Rb1, may result in
decreased or complete absence of protein expression by immu-
nohistochemistry. Rb1 is an important tumor suppressor gene
regulating the cell cycle, and mutation or loss causes
unchecked cell proliferation. CDKN2A inhibits phosphoryla-
tion of RB1, thereby preventing cell cycle progression. Muta-
tion in either of these tumor suppressor genes results in
decreased or loss of expression of their protein products in can-
cer cells. PCR analysis to detect loss of one or both alleles or
gene sequencing should be used to identify the specific muta-
SIGNIFICANCE OF GENETIC ALTERATIONS IN
TUMOR INITIATION AND PROGRESSION IN RODENTS
As models of environmental and occupational exposure to
carcinogens, investigation of the underlying mechanisms of
tumorigenesis in rodents can yield valuable information as to
the molecular events taking place in the progression of lesions
from preneoplastic lesions to neoplasia. Although metastasis is
842 HOENERHOFF ET AL.TOXICOLOGIC PATHOLOGY
difficult to study in chemically induced models of neoplasia
since it is a relatively infrequent and time-dependent occur-
rence, it has been extensively studied in genetically engineered
mouse models. Knowledge of the genetic lesions occurring
from one stage to the next can provide important information
regarding the potential pathogenesis of these lesions in humans
that are potentially exposed to such compounds. The genetic
mutations that occur in rodent models often follow a distinct
progression in the development from benign tumors to malig-
nant cancers. Chemically induced and transgenic rodent mod-
els are frequently used to model preneoplastic and neoplastic
lesions that result from genetic alterations in humans, and such
models give insight into mechanisms and potential treatment or
provide information on current applications of diagnostic mod-
alities for use in human patients. Many of the genetic altera-
tions we have identified in chemically induced rodent models
of cancer mimic changes present in human cancer in terms of
stages of initiation and progression from premalignancy to
Initiation: Several of our chemically induced mouse models of
lung cancer have alterations in the Kras and Tp53 genes, two
genes commonly mutated in human lung cancer. Mutations
in Kras are believed to be an early initiating event in lung car-
cinogenesis in humans (Spivack et al. 1997; Westra, Slebos,
et al. 1993) and mice (Donnelly et al. 1996; Horio et al.
1996; Spivack et al. 1997; Wakamatsu et al. 2007). Similarly,
in chemically induced rodent liver tumor models as well as
human liver tumors, Ras mutation is associated with early
events in tumorigenesis. For example, increased levels of
HRAS, NRAS, and KRAS have been reported in some pre-
neoplastic liver lesions in humans (Coleman 2003), and
activated Kras and Hras oncogenes have been detected in
chemically induced mouse liver tumors (Maronpot et al.
1995; Reynolds et al. 1988). Beta-catenin gene mutation is
another important event in mouse and human hepatic tumors
that has been shown to be an early initiating event in chemi-
cally induced mouse (Devereux et al. 1999) and rat (Yamada
et al. 1999) hepatocellular carcinogenesis. Expression of insu-
lin growth factor II (IGFII) and transforming growth factor
alpha (TGFA) are associated with preneoplastic lesions in
humans (Thorgeirsson and Grisham 2002) and contribute to the
formation of liver tumors in mouse models, including IGFII
expression during early stages of liver carcinogenesis (Lahm
et al.2002).Although dysregulation of b-catenin andWNT sig-
naling most often follows mutation of the APC gene as an initi-
ating event in human colon cancer, mutation of the b-catenin
gene as a primary initiating event also occurs in a subset of
colon cancers in humans (Fodde, Smits, and Clevers 2001;
Mirabelli-Primdahl et al. 1999), as well as a chemically
induced mouse model of large intestinal (cecal) carcinoma
(Sills et al. 2004). In addition, Kras activating mutations have
been shown to be an early initiating event in a rat model of che-
mically induced colon carcinogenesis (Jacoby et al. 1991;
Rosenberg, Giardina, and Tanaka 2009).
Progression: Mutations in TP53 are frequently observed in
human lung adenocarcinomas (Hwang et al. 2003; Westra,
Offerhaus, et al. 1993), as well as mouse lung tumors
(Wakamatsu et al. 2007) and can act synergistically with Kras
mutation in the development of certain cancers in mice and
humans (Fisher et al. 2001; Halevy, Michalovitz, and Oren
1990; Pierceall et al. 1991; Wakamatsu et al. 2007; Wang
et al. 2006). Mutation of TP53 occurs late in the course of dis-
ease and plays an important role in progression to malignancy
in mice (Horio et al. 1996; Jackson et al. 2005; Wakamatsu
et al. 2007) and is associated with a poor prognosis in human
patients with non-small-cell lung cancer (Huang et al. 1998).
In the liver, TP53 mutation is associated with later stages in
aflatoxin-induced hepatocellular carcinogenesis (Liu, Lin, and
Ng 1996) and advanced stages of tumorigenesis in humans
(Hsu et al. 1993).
Several other studies have investigated the role of distinct
genomic aberrations in the progression from premalignant to
neoplastic lesions in chemically induced rodent models of
human liver (Longato et al. 2009; Tward et al. 2007), lung
(Hutt et al. 2005; Malkinson 1992), colon (Yamada and Mori
2007), mammary gland (Medina 2008; Russo and Russo
1996), skin (Balmain and Pragnell 1983; Brown, Buchmann,
and Balmain 1990), and other cancers, and the impact of
genetically engineered mouse models has continued to increase
the understanding of genetic alterations and their effect on car-
cinogenesis. Thus, the occurrence of these mutations in the
mouse suggests that chemically induced mouse models of car-
cinogenesis can in many ways model human tumorigenesis.
IMPACT ON RISK ASSESSMENT FOR HUMAN HEALTH
Several methods are used to identify environmental or occu-
pational hazards that pose significant carcinogenic risks to
humans, including in vitro mutagenesis assays and in vivo
long-term carcinogenicity assays in rodents, with classification
of a compound as a hazard based predominantly on the latter
(Reynolds et al. 1988). Incidence of neoplasia in a rodent spe-
cies as a result of chemical exposure provides information on
risk assessment for human health; however, it is difficult to
determine whether an exposure in humans will elicit a similar
response, since hazard identification and risk assessment
requires the presumption of similarity between rodents and
humans (Holsapple et al. 2006). This is challenging due to sev-
eral reasons. First, the metabolic activity of mice and rats may
differ significantly not only between one another but also from
that of other species such as human. Second, there are signifi-
cant differences in tumor susceptibility between strains of mice
as well as rats. Finally, doses administered to rats and mice are
often a greatermagnitude than thatof whathumans are exposed
to. Therefore, often it is difficult to extrapolate the findings in
rodents exposed to chemical carcinogens tothat of whatmay be
predicted in humans. What does this mean for human risk
assessment? The induction of a tumor response in rodents as
a result of exposure to a compound alone is becoming insuffi-
cient as a means of prediction of human disease. While this
Vol. 37, No. 7, 2009 MECHANISMS OF CANCER IN NTP RODENT BIOASSAYS843
tells us a certain compound is a rodent carcinogen, it does not
definitively confirm that the same effect will be seen in humans
at the same site or by a similar mechanism. The development of
molecular investigation into the underlying genetic and epige-
netic mechanisms of carcinogenesis is providing a more
detailed and definitive explanation of why cancer occurs in
these models. As a result, scientists and regulators may com-
pare genetic and epigenetic mechanisms in rodent models to
those known to occur in human cancers in order to develop a
more predictive model of chemically induced carcinogenesis
in humans. These approaches will continue to enable regulators
to make more definitive and confident decisions regarding
human exposure to certain compounds and may remove some
of the uncertainty related to species and dose extrapolation of
human health risk from rodent carcinogenicity data. Further-
more, the investigation into the molecular pathogenesis of
these models should become a mainstay in decision making
regarding the prediction of human health risks and regulation
of various occupational and environmental compounds.
CONCLUSIONS AND SUMMARY
The use of the mouse and rat as models of chemically
induced carcinogenesis, both environmental and occupational,
has provided evidence that human exposure to these chemicals
may pose a significant health risk for developing cancer. These
animal models provide us with tools to decipher the molecular
mechanisms that may be at play in the genesis of human cancer
and, therefore, may be extremely valuable to the understanding
of the underlying genetic and nongenetic causes of human can-
cer. Our group has investigated numerous compounds that pose
a potential human cancer risk. From these studies, we have
identified several genetic events, and in future studies, we plan
to examine epigenetic pathways responsible for the develop-
ment of different types of chemically induced tumors in the
mouse and rat. Using mutation analysis, gene expression stud-
ies, immunohistochemistry, and other allied research tech-
niques, we have identified several factors in the rodent
models that are responsible for the generation of cancer. This
research has been important in furthering our understanding
of the mechanisms of chemically induced carcinogenesis and
the potential risks that certain environmental, occupational, and
food-related chemical compounds may have on human health.
Our studies of genetic and epigenetic mechanisms of cancer
will continue to contribute to our understanding of the molecular
pathogenesis of cancer and in the evaluation of environmental
risks. By understanding the molecular pathogenesis of cancer,
we can relate oncogenic or epigenetic events occurring in the
rodent to changes that occur in human cancer and make conclu-
treatment of various different types of human cancer.
We thank the many toxicologists, pathologists, and NTP/
NIEHS staff who contributed to this research. This research
was supported by the Intramural Research Program of the NIH,
National Institute of Environmental Health Sciences.
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