The Toxicologist - Society of Toxicology

mkd. Cyp2b10 was the strongest indicator of exposure and the overall response indicated
increased expression changes of genes whose protein products have functions
in cell proliferation and xenobiotic responses. Beyond a predominant metabolic
transcriptional response affecting fatty acid and xenobiotic metabolism, other
gene expression associated responses were NRF2-mediated oxidative stress, inflammation,
numerous cytokine, and apoptotic pathways. Together, these data inform
dose, time, and sex effects underlying PB hepatotoxicity in rodents and the mode of
action leading to tumor promotion.
2640 OXIDATIVE DNA DAMAGE AND DNA BINDING
INDUCED BY 2, 2-BIS (BROMOMETHYL)-1, 3-
PROANEDIOL IN HUMAN BLADDER CELLS:
POSSIBLE DUAL MECHANISMS IMPLICATED IN ITS
CARCINOGENICITY.
W. Kong, R. K. Kuester, A. Gallegos and I. Sipes. Pharmacology, University of
Arizona, Tucson, AZ.
2, 2-Bis (bromomethyl)-1, 3-propanediol (BMP) is a flame retardant found in urethane
foams and polyester resins. In a two year dietary study, BMP caused neoplastic
lesions at multiple sites including the urinary bladder of rats and mice. The
mechanism of its carcinogenic effect is unknown. In this in vitro study, using SV-40
immortalized human uroepithelial cells (UROsta), binding of BMP to DNA and
BMP induced DNA damage were investigated. DNA binding levels were measured
at various time points between 1 and 24 h of incubation of UROsta cells with 14C-
BMP (10 μM). The effects of time (1-24 h) and concentration (5-100 μM) on
DNA strand breaks were assessed via the standard alkaline comet assay. Binding of
total BMP to DNA increased from 3 to 38 pmol per mg DNA over the incubation
time (resistant to RNAase and proteinase digestion and phenol-chloroform extraction).
BMP significantly increased the extent of DNA strand breaks at 1 and 3h of
incubation. However, this DNA damage returned to basal level after 6h. Because
the induction of strand breaks preceded the maximum binding, a potential role for
BMP associated oxidative damage to DNA was investigated. Cell lysates were incubated
with a lesion specific endonuclease, human 8-hydroxyguanine DNA-glycosylase
(hOGG1) before the alkaline comet assay to reveal oxidized guanine as strand
breaks. Also, intracellular reactive oxygen species (ROS) were measured by a ROS
sensing fluorescence probe, H2DCFDA. An increase in strand beaks upon incubation
with hOGG1 was observed in the modified comet assay at 1h following BMP
exposure. Furthermore, evidence for BMP (10, 25,100 μM) associated oxidative
stress was shown as an elevation of intracellular ROS generation within 1h. These
results demonstrate that in UROsta cells BMP binds to DNA and also induces
DNA strand breaks, some of which result from oxidative DNA lesions. Both of
these events, DNA binding and oxidative DNA damage may, in part, contribute to
BMP carcinogenicity.
2641 TRICHLOROETHYLENE- AND
TETRACHLOROETHYLENE-INDUCED RENAL
CARCINOGENESIS: INSIGHTS FROM HUMAN,
ANIMAL BIOASSAY, AND MECHANISTIC STUDIES.
M. R. Gwinn, K. Z. Guyton, J. Jinot, C. S. Scott, K. A. Hogan and W. A.
Chiu. U.S. EPA, Washington, DC.
A review was conducted of epidemiologic, animal bioassay and mechanistic data for
insights regarding the effects of trichloroethylene (TCE) and tetrachloroethylene
(PCE) on kidney toxicity and carcinogenicity. Numerous independent epidemiologic
studies have associated TCE exposure with elevated kidney cancer risk and statistically
significant exposure-response trends, and the consistency of the findings
suggests a causal relationship between TCE exposure and kidney cancer. For PCE,
the epidemiologic evidence of kidney cancer is more limited, with most studies examining
job or occupational title. The rodent bioassay database includes multiple
lifetime studies reporting small increases in renal tubule tumors, including very rare
malignant adenocarcinomas, primarily in male rats following inhalation or oral exposure
to TCE or PCE. Renal toxicity but not cancer is reported in mice. Results of
in vivo and in vitro studies support a prominent role for metabolites derived from
the GSH conjugation pathway of TCE or PCE metabolism in renal toxicity and tumorigenicity.
This includes the demonstrated in vitro mutagenicity of the GSH-derived
PCE and TCE metabolites, or of both parent compounds under conditions
that would generate these metabolites, and evidence of their generation in the kidney
following PCE or TCE exposure. The role of GSH conjugates is further supported
by recent epidemiologic evidence of elevated kidney cancer risk in TCE-exposed
subjects with at least one intact GSTT1 allele but not among subjects with
two deleted alleles. While the mechanisms of PCE- and TCE-induced renal carcinogenesis
have not been fully elucidated, this review of the available evidence sup-
566 SOT 2011 ANNUAL MEETING
ports the contribution of these mutagenic metabolites to the development of the
kidney tumors for either compound. Some evidence supports a role of cytotoxicity
leading to compensatory cellular proliferation in renal carcinogenesis, either together
with or independent of mutagenicity. This abstract does not represent EPA
policy.
2642 COMPARISON OF AFLATOXIN-DNA ADDUCT LEVELS
IN MALE AND FEMALE INFANT B6C3F1 MICE.
L. L. Woo 1 , P.A.Egner 2 , C. Belanger 1 , R. Wattanawaraporn 1 ,L.Trudel 1 ,G. N.
Wogan 1, 3 ,J. D. Groopman 2 , R.G.Croy 1, 3 and J. M. Essigmann 1, 3 . 1 Biological
Engineering, Massachusetts Institute of Technology, Cambridge, MA, 2 Environmental
Health Sciences, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD
and 3 Chemistry, Massachusetts Institute of Technology, Cambridge, MA.
The infant mouse model is useful for identification of developmental factors responsible
for the age and gender differences in sensitivity towards AFB 1 and other
carcinogens. Male infant B6C3F1 mice are highly sensitive to the hepatocarcinogenic
effects of aflatoxin B 1 (AFB 1 ), while infant females are refractory. A multidose
regimen of AFB 1 beginning at 4 days of age produces hepatocellular carcinoma
in >90% of males at 18 months; no tumors are observed in females at that
time point. Adult mice of both genders, unlike infant males, are resistant to AFB 1 ’s
carcinogenic effect. AFB 1 is metabolized to the 8,9-epoxide which covalently modifies
DNA, forming mutagenic AFB 1 -N7-Gua and AFB 1 -FAPY adducts. We investigated
whether male and female infant mice are at similar risk of genetic damage
from AFB 1 . AFB 1 adducts in liver DNA were measured 24 h following administration
of either a single 6 μg/g dose on day 4 or as 3 x 2 μg/g doses administered on
days 4, 7 and 10 of age. AFB 1 adducts were released from isolated DNA by acid hydrolysis
and quantified by HPLC-MS/MS using AFB 1 - 15 N 5 -Gua internal standards.
Following a single 6 μg/g AFB 1 dose, 3.4± 1.7 and 2.8±0.90 AFB 1 -N7-Gua
adducts/10 6 DNA bases were found in male and female mouse livers, respectively.
Twenty-four h after the final 2 μg/g dose, levels of AFB 1 -N7-Gua were much lower
at 0.37±0.21 adducts/10 6 bases in males and 0.40±0.20 adducts/10 6 bases in females.
AFB 1 -FAPY adducts were at higher levels in the multi-dose animals; 3.6
±1.9 AFB 1 -FAPY adducts/10 6 bases in males and 2.5±1.2 AFB 1 -FAPY adducts/10 6
bases in females. These results indicate that similar amounts of AFB 1 -DNA adducts
are formed in male and female infant B6C3F1 mice. Thus, both genders appear to
be at a similar risk of mutations from covalent AFB 1 adducts formed in liver DNA.
2643 NO LUNG TOXICITY FROM STYRENE IN CYP2F2
KNOCKOUT MICE.
G. Cruzan 1 , J. S. Bus 2 , X. Ding 3 , J. A. Hotchkiss 2 , J. R. Harkema 4 and R.
Gingell 5 . 1 ToxWorks, Bridgeton, NJ, 2 Toxicology and Environmental Research and
Consulting, Dow Chemical Company, Midland, MI, 3 New York State Department of
Health, Wadsworth Center for Labs. and Research, State University of New York at
Albany, Albany, NY, 4 Pathobiology and Diagnostic Investigation, Michigan State
University, East Lansing, MI and 5 Environmental and Product Health, Shell Oil
Company, Houston, TX.
Styrene induces lung tumors in mice, but not in rats. We hypothesized that the
mouse lung tumors were mediated by mouse specific CYP2F2 generated cytotoxic,
ring-oxidized metabolite(s) producing repeated localized injury to Clara cells, the
site of CYP2F2 metabolism and the tumorigenic response. This hypothesis was
consistent with previous data indicating that styrene lung toxicity was not attenuated
in CYP2E1 knockout mice (2E1 produces styrene-7,8-oxide). To test our hypothesis
we developed CYP2F2-/- (KO) mice on a C57BL/6 background. The
2F2-KO mouse was produced by replacing the third coding exon with a neomycin
resistance gene followed by homologous recombination in C57BL/6-derived
Bruce4 mouse embryonic stem cells. Male WT and KO mice received 0 or 400
mg/kg/day styrene in corn oil by gavage for 5 days (n=7/group). WT mice treated
with 400 mg/kg styrene displayed mild atrophy of the bronchiolar epithelium due
to necrosis and exfoliation of Clara cells concurrent with areas of epithelial regeneration.
The cumulative BrdU-labeling of S-phase cells (LI) was markedly increased
in both proximal (46.8±7.9%) and terminal bronchioles (62.4± 2.7%) compared
to WT control mice (2.2±0.5% and 4.4±0.9%, respectively). No evidence of Clara
cell toxicity was observed in the pulmonary airways of 400 mg/kg-treated KO mice.
The cumulative LI was not increased in proximal (1.1±0.3%) or terminal bronchioles
(2.2±0.7%) compared to KO control mice (3.2±0.6% and 2.0±0.7%, respectively).
This study clearly demonstrates that styrene toxicity in the mouse lung is
dependent on metabolism by CYP2F2, and that the mouse tumor response may
not be relevant to human risk in that human lung expresses CYP2F1, an isoform
not suspected of catalyzing significant styrene metabolism.