The Toxicologist - Society of Toxicology

various pathological situations, some studies have proposed them as potential therapeutic
agents in medical applications. Thus, the potential risks of uses of these nitroxides
need to be addressed. In this study, we investigated the cytotoxicity and
mutagenicity of Tempo and Tempol using the mouse lymphoma assay. In the absence
of metabolic activation, the L5178Y/Tk+/—3.7.2C mouse lymphoma cells
treated with 3 mM Tempo showed significant cytotoxicity and marginal mutagenicity.
In the presence of S9, the treatment of cells with Tempo at concentrations
of 1-2 mM resulted in a decrease of the relative total growth (RTG) and an increase
of mutant frequency. Treatment of cells with Tempol at concentrations of 2-10 mM
with or without S9 resulted in dose-related increases of both cytotoxicity and mutagenicity.
Within the dose range tested, oxidative stress induced by Tempo and
Tempol was detected in the mouse lymphoma cells. Both compounds showed significantly
reduced glutathione (GSH) levels in a dose-related manner in the all
treatment groups. In addition, Tempo resulted in approximately a 1.5-fold increase
of the reactive oxygen species (ROS) in all treatment doses. To explore the underlying
mutagenic mechanism, we examined the loss of heterozygosity (LOH) at four
microsatellite loci spanning the entire chromosome 11 for the Tk mutants induced
by Tempo and Tempol. The mutational spectra induced by both compounds, in
which the majority of mutants showed LOH at the Tk locus only (i.e., chromosome
damage less than 30 centimorgan), were distinctly different from the vehicle
control. These results suggest that Tempo and Tempol are mutagenic in mouse lymphoma
cells with a clastogenic mode-of-action.
1408 GENOTOXICITY OF 3, 5-DIMETHYLANILINE AND ITS
METABOLITES.
M. Chao, L. J. Trudel and G. N. Wogan. Biological Engineering, Massachusetts
Institute of Technology, Cambridge, MA.
Epidemiological studies have demonstrated extensive human exposure to the
monocyclic aromatic amine, 3,5-dimethylaniline (3,5-DMA). Although some aromatic
amines such as 4-aminobiphenyl are well-documented risk factors for bladder
cancer, little is known about the genotoxicity and potential carcinogenic risk created
by 3,5-DMA exposure. We therefore are investigating the cytotoxicity and mutagenicity
of 3,5-DMA and its metabolites N-hydroxyl-3,5-dimethylaniline (N-
OH-3,5-DMA) and 3,5-dimethylaminophenol (3,5-DMAP) in Chinese hamster
ovary (CHO) cells. Cytotoxicity and aprt mutagenicity induced by these chemicals
in nucleotide repair-proficient (RP) AA8 and repair-deficient (RD) UV5 cell lines.
These cells do not express significant levels of phase I and II enzymes necessary for
xenobiotic activation. Results to date show that the parental chemical, 3,5-DMA,
as well as the N-OH-3,5-DMA and 3,5-DMAP derivatives all caused dose-dependent
decreases in cell survival, but with very different potencies: 3,5-DMAP had the
highest potency; N-OH-DMA intermediate; and 3,5-DMA lowest. No aprt mutations
were induced by these treatments, even at highly toxic doses. It was noted
however, that exposure to these compounds induced dose-dependent production of
ROS, as determined by carboxyl-H2DCF-DA fluorescence. To assess the contributions
of phase I and II enzymes to the toxicity of these alkylanilines, experiments as
described above were conducted in isogenic AA8 and UV5 cells genetically modified
to express human cytochrome P4501A2 (CYP1A2) and N-acetyltransferase-2
(NAT2). Treatment with the alkylanilines again caused dose-dependent cell killing,
with 3,5-DMAP being most potent, N-hydroxy-3,5-DMA intermediate and 3,5-
DMA least potent. All three compounds also induced substantial increases in aprt
mutation frequency at dose levels reflecting their cytotoxic potencies. In summary,
these results indicate that 3,5-DMA and its metabolites are cytotoxic to the cells
and require CYP1A2 and NAT2 to exert their mutagenic effects.
1409 COMPARISON OF THE BACTERIAL MUTAGENICITY
OF MAINSTREAM WHOLE SMOKE FROM
CIGARETTES WITH DIFFERENT LEVELS OF
MENTHOL.
R. Leverette. Lorillard, Greensboro, NC.
Menthol is widely used in the pharmaceutical, cosmetic, food and tobacco industries
and is generally regarded as safe (GRAS) for these applications. This study was
conducted to compare the mutagenicity of mainstream whole-smoke (WS) and wet
total particulate matter (WTPM) from two sets of experimental cigarettes to determine
if menthol has an effect on this endpoint. The first pair of experimental cigarettes
(delivering ~6mg WTPM/cig) included a mentholated (0.6% w/w menthol)
and a comparable non-mentholated control. The second set consisted of four experimental
cigarettes (delivering ~9mg WTPM/cig) with increasing levels of menthol
(0.1% - 0.7% w/w) and a non-mentholated control. Within each set, the cigarettes
were comparable in construction, composition, and WTPM deliveries with
added menthol spanning typical user levels. All cigarettes were smoked under ISO
puff profile (35mL volume, 2 second duration and 1 minute interval) on a VIT-
ROCELL® VC10 smoking robot, with WTPM being pad collected and extracted
302 SOT 2011 ANNUAL MEETING
in dimethylsulfoxide. TA98 and TA100 were exposed to WS or WTPM with metabolic
activation (S9+) utilizing either the VITROCELL® Ames exposure modules
and whole-smoke (WS) dilution system or the preincubation method (WTPM).
The WS specific activity (revertants/ug) from the first pair of sample cigarettes
showed the mentholated cigarette had significantly lower activity then the nonmentholated
control (TA98, p = 0.0027; TA100, p = 0.0297). For the second set of
cigarettes, only small differences in WS activities were observed, with a trend of decreasing
activity with increasing menthol that was not statistically significant. No
differences were detected for WTPM specific activities. No significant differences
in WS activities occurred when cigarettes were removed from packs and conditioned
(18 hours, 23°C, 60% RH) prior to smoking. With no changes in smoke delivery
or cytotoxicity and with the reduction of menthol from the tobacco during
conditioning, these results suggest menthol may have a role in the observed reduction
of WS Ames activity.
1410 A NOVEL IN VITRO SYSTEM FOR THE
TOXICOLOGICAL EVALUATION OF GENOTOXIC
COMPOUNDS.
S. O. Mueller, K. Boehme, Y. Dietz and P. G. Hewitt. Toxicology, Merck KGaA,
Darmstadt, Germany.
Genotoxicity is an important issue during pharmaceutical development and chemical
risk assessment. In vivo carcinogenicity studies are animal, time and cost-intensive
and in vitro tests have relatively low specificity. Furthermore, economic and animal
welfare aspects, in particular within the scope of REACH, indicate the
importance of new in vitro tools. Previous studies have proven the suitability of toxicogenomic
approaches for the predictive classification of genotoxicants in vivo, but
in vitro prediction is still a challenge.
This study aimed to develop a new genomics-based test system for mutagens and
promutagens in vitro using HepG2 cells. Due to their limited metabolic capacity
we established a combined system of HepG2 cells and a metabolic activation system
(MAS – rat liver S9) for promutagen testing. P53 activation in nuclear protein
extracts of HepG2 cells served as surrogate marker for compound-related genotoxicity
and has confirmed the functionality of the MAS-HepG2 system. Global gene
expression changes were quantified 24h and 48h after treatment with (pro-)genotoxic
(cyclophosphamide, dimethylbenz[a]¬anthracene, Aflatoxin B1, actinomycin
D, methyl methanesulfonate, etoposide) and non-genotoxic (metformin, theophylline)
compounds using Illumina BeadChip arrays. Differential gene regulations
between both compound classes were identified by gene Ranking with an ANOVA
for group separation followed by support vector machine algorithm for classifier
calculation. The best classification was achieved using the 91 top-scored genes. The
predicitvity was evaluated by k-fold cross validation, showing an appropriate group
allocation according to the specified annotation. Thereafter, the classifier built was
used to predict the toxicity of diethylnitrosamine correctly.
The 91 genes identified are potential candidates for the mechanistic characterization
and identification of new genotoxicants. Combined with other genotoxicity
endpoints as well as the inclusion of metabolic activation might enable a holistic
screening system for a broad range of potentially genotoxic compounds.
1411 COMPARATIVE MUTAGENICITY OF
CYCLOPHOSPHAMIDE IN F344 MALE RATS USING
THE PIG-A AND HPRT ASSAYS.
V. N. Dobrovolsky, J. G. Shaddock, M. Pearce and R. H. Heflich. NCTR,
Jefferson, AR. Sponsor: B. Parsons.
Cyclophosphamide (CP) is a bifunctional alkylating agent that forms interstrand
crosslinks in double-stranded DNA. Even though it is a potent clastogen and
known human carcinogen, CP is frequently used for cancer chemotherapy and to a
lesser degree for treatment of autoimmune diseases. In the present study, we administered
single i.p. doses of 10, 40 and 80 mg/kg CP to male F344 rats and evaluated
its ability to induce gene mutation in two assays that employ X-linked endogenous
reporter genes, the red blood cell (RBC) Pig-a assay and the lymphocyte
Hprt assay; the reticulocyte (RET) micronucleus assay for clastogenicity/aneugenicity
was performed for comparison. Micronucleated (MN) RETs and percent
RETs (as a measure of cytotoxicity) were assayed by flow cytometry in peripheral
blood 48 hr posttreatment, while the Pig-a assay (measuring the frequency of
CD59-deficient peripheral RBCs) was conducted using flow cytometry at 1, 2, 3,
4, 6, 8, 9, 12 and 16 weeks posttreatment, and the frequency of 6-thioguanine-resistant
(Hprt mutant) spleen T-cells was determined in clonal assays at 3, 6, 9 and
16 weeks. CP treatment produced a strong short-term suppression of erythropoiesis
and 10-fold increase in MN RET frequency (0.07% in untreated controls to ca.
0.8% in treated animals). The RBC Pig-a mutant frequency was marginally ele-