The Toxicologist - Society of Toxicology

1108 STYRENE-INDUCED TOXIC EFFECTS IN ALDH2
KNOCKOUT MICE.
R. Wang 1 , K. Ohtani 1 , L. J. McGarrity 2 , Z. Weng 1 and N. Mei 2 . 1 Japan
National Institute of Occupational Safety and Health, Kawasaki, Japan and
2
National Center for Toxicological Research/U.S. FDA, Jefferson, AR.
Styrene is an important chemical widely used in manufacturing plastics and synthetic
rubber. Styrene exposure has been reported to cause DNA damage as shown
by the increased DNA adducts and chromosomal aberrations. This effect has been
correlated with the genetic polymorphisms of metabolizing enzymes such as CYPs
and GSTs, and DNA repair enzymes. Aldehyde dehydrogenase 2 (ALDH2) is also
involved in the metabolism of styrene, but the enzyme activity is deficient in about
40 percent of East Asian population due to a mutant allele of the gene encoding the
enzyme. In this study, we used ALDH2 gene knockout (KO) mice to detect any difference
in styrene induced effects such as micronuclei and other endpoints compared
with those in wild type (WT) mice. Male and female mice were administrated
with styrene at 0, 100, 400 and 800 mg/kg, p.o., per day, 5 days per week, for
4 consecutive weeks. Peripheral blood was sampled 24 hr after the last treatment
and the micronucleus in the reticulocytes was detected by a flow cytometric
method using kit from Litron Laboratories. The background of the frequency of
micronucleated reticulocytes (MN-RETs) was higher in KO mice than in the WT
mice for both male and female. The percentage of the MN-RETs was significantly
increased by approximately 40% in the male mice treated with the high dose of
styrene and this effect was not observed in the male WT mice. No evident change
in the groups of low and middle doses was detected. In female KO mice, the percentage
of MN-RETs showed a dose-dependent induction with styrene treatment,
with statistically significant increase in the middle and high dose groups as compared
with the control. Again this toxic effect was only found in the KO, but not
WT mice. These results suggest that ALDH2 polymorphisms is another genetic factor
that may modify the toxic effects of styrene and may be used as a biomarker of
susceptibility to styrene exposure.
1109 EVALUATION OF L-MENTHONE IN AN IN VIVO
MOUSE MICRONUCLEUS TEST.
J. Scognamiglio, V. T. Politano and A. Api. Research Institute for Fragrance
Materials, Woodcliff Lake, NJ.
A mouse micronucleus test was conducted on l-menthone (CAS # 14073-97-3), a
widely used fragrance ingredient, to determine its potential to induce micronuclei
in the polychromatic erythrocytes (PCEs) in the bone marrow of the mouse to evaluate
clastogenic potential. In the dose range-finding phase, 2000 mg/kg bodyweight
was determined suitable as it was the maximum guideline-recommended
dose. In the definitive study, l-menthone was then administered orally at dose levels
of 500, 1000 or 2000 mg/kg in corn oil to NMRI mice (5/sex/dose). The control
groups were treated with the vehicle, corn oil, or cyclophosphamide (CPA) at 40
mg/kg as the positive control. The control and test article were both orally administered
at a volume of 10 mL/kg body weight. Bone marrow cells were harvested 24
and 48 hours after treatment. At least 2000 PCEs per animal were scored for micronuclei.
The number of PCEs showed no statistically significant increases when
compared to that of the vehicle control. Therefore, l-menthone administered at
doses up to 2000 mg/kg did not induce a statistically significant increase in micronuclei
in either male or female mice and is not genotoxic.
1110 EVALUATION OF ISOAMYL ALCOHOL IN AN IN VIVO
MOUSE MICRONUCLEUS TEST.
D. McGinty, V. T. Politano and A. Api. Research Institute for Fragrance Materials,
Inc., Woodcliff Lake, NJ.
Isoamyl alcohol, a widely used fragrance ingredient, was tested in an in vivo mouse
micronucleus assay to evaluate the clastogenic potential as measured by its ability to
induce micronucleated polychromatic erythrocytes in mouse bone marrow. This
test is a short-term in vivo cytogenetic assay for detecting agents that induce chromosomal
breakage and spindle malfunction. In the dose range-finding phase, 2000
mg/kg bodyweight was determined suitable as it was the maximum guideline-recommended
dose. The definitive micronucleus study consisted of several groups,
each containing 5 male and 5 female NMRI mice. The control groups were treated
with the vehicle, corn oil, or cyclophosphamide (CPA) at 40 mg/kg as the positive
control. Isoamyl alcohol was tested at dosages of 500, 1000, or 2000 mg/kg. Both
the test and control articles were administered via gavage at a dose volume of 10
ml/kg body weight. Bone marrow cells (polychromatic erythrocytes, PCEs) were
collected 24 and 48h after the treatment and examined microscopically for the
presence of micronuclei. At least 2000 PCEs per animal were scored for micronuclei.
There was no statistically significant increase in the incidence of micronucleated
polychromatic erythrocytes observed in test article treated groups relative to respective
vehicle control groups. Therefore, isoamyl alcohol at doses up to and including
2000 mg/kg did not induce a significant increase in micronuclei in either
male or female NMRI mice and may be concluded as negative in the mouse micronucleus
assay.
1111 AN IN VIVO MOUSE MICRONUCLEUS TEST WITH
GAMMA-NONALACTONE.
C. Letizia, A. Api and V. Politano. RIFM, Woodcliff Lake, NJ.
An in vivo mammalian erythrocyte micronucleus test was conducted to evaluate the
potential of gamma-nonalactone (CAS No. 104-61-0), a widely used fragrance material,
to induce micronuclei in polychromatic erythrocytes (PCEs) in the bone
marrow of the mouse. The study was conducted in compliance with GLP and
OECD Test Guideline 474 (Mammalian Erythrocyte Micronucleus Test). Male
and female NMRI mice (5/sex/dose) were administered gamma-nonalactone orally
at dose levels of 500, 1000 or 2000 mg/kg body weight in corn oil at a dose volume
of 10 ml/kg body weight and sacrificed at 24 or 48 hours after dosing. Control
groups received the vehicle alone or cyclophosphamide (40 mg/kg body weight) as
a positive control. PCEs were collected at 24 and 48 hours after administration and
examined microscopically for the presence of micronuclei. At least 2000 PCEs per
animal were scored for micronuclei. The number of PCEs was not substantially decreased
in gamma-nonalactone treated groups compared to the mean value of PCEs
of the vehicle control group indicating that gamma-nonalactone did not produce
any cytotoxic effects in the bone marrow. There was no significant increase in the
incidence of micronucleated PCEs in gamma-nonalactone treated groups relative to
their respective vehicle controls in either male or female mice, regardless of dose
level or bone marrow collection time. The mean values of micronuclei observed in
the gamma-nonalactone treated groups were below or near vehicle control values.
Cyclophosphamide induced a statistically significant increase in micronucleated
PCEs in both male and female mice. It was concluded that under the conditions of
the test, gamma-nonalactone was not genotoxic.
1112 DOSE-RESPONSE CHARACTERIZATION OF VINYL
ACETATE AND ACETALDEHYDE-INDUCED
MICRONUCLEI IN HUMAN TK6 CELLS.
R. Budinsky 1 , B. Gollapudi 1 , R. J. Albertini 2 , R. Valentine 3 , M. Stavanja 4 , J.
Teeguarden 5 , R. J. Fensterheim 6 and L. Recio 7 . 1 The Dow Chemical Company,
Midland, MI, 2 The University of Vermont, Burlington, VT, 3 DuPont Haskell Global
Centers for Health and Environmental Sciences, Newark, DE, 4 Celanese
International Corporation, Dallas, TX, 5 Battelle, Pacific Northwest Division,
Richland, WA, 6 RegNet Environmental Services, Washington, DC and 7 ILS, Inc.,
Research Triangle Park, NC.
Chronic vinyl acetate monomer (VAM) inhalation induces rat nasal tumors when
concentrations exceed 200 ppm. VAM’s metabolite, acetaldehyde (AA), is hypothesized
to be the genotoxic metabolite produced by tissue carboxylesterase. AA metabolism
to acetic acid, via acetaldehyde dehydrogenase, leads to acetic acid accumulation,
reduced intracellular pH, cytotoxicity, and regenerative cell proliferation.
To shed more light on VAM-induced nasal tumors, an investigation was undertaken
to obtain robust dose-response information on the induction of cytogenetic
damage by VAM and AA in human TK6 cell cultures. A flow cytometry-based micronucleus
assay was utilized to enhance the quantitative sensitivity of cytogenetic
damage. Micronucleus induction was accompanied by significant cytotoxicity. Five
different dose-response modeling methods provided threshold estimates of 0.01 to
0.75 mM and 0.05 to 0.14 mM for VAM and AA, respectively. Physiologicallybased
toxicokinetic model estimates of VAM and AA concentrations in regions associated
with rodent nasal tumors are substantially lower than the estimated in-vitro
micronuclei thresholds. Additional investigation is needed to evaluate the relevance
of the in-vitro to in-vivo concentration comparisons. These results support the
threshold nature of VAM-induced rat nasal tumors that are hypothesized to result
from exceeding otherwise efficient metabolism of AA and consequent accumulation
in AA or decreases in cellular pH when proton production exceeds the cellular
buffering capacity.
1113 AUTOMATED MICRONUCLEUS/DNA CONTENT
ASSAY BY QUANTITATIVE IMAGING CYTOMETRY.
E. Holden, M. Henriksen, E. Luther and S. Baldwin. CompuCyte Corporation,
Westwood, MA. Sponsor: P. Narayanan.
The in vitro micronucleus (MN) assay is a well established test of compound genotoxic
effects. We describe the validation of multi parameter in-vitro micronucleus
assay with simultaneous measurement of cell counts and DNA content. Methods:
SOT 2010 ANNUAL MEETING 237