The Toxicologist - Society of Toxicology

1416 PIG-A MUTATION AND MICRONUCLEATED
RETICULOCYTE ASSAYS DISCRIMINATE THE
MUTAGENIC/NON-MUTAGENIC PAIR:
BENZO[A]PYRENE/PYRENE.
D. Torous 1 , J. MacGregor 2 , S. Phonethepswath 1 , P. Weller 1 , J. Bemis 1 and S.
Dertinger 1 . 1 Litron Laboratories, Rochester, NY and 2 Toxicology Consulting Services,
Arnold, MD.
Integration of genotoxicity endpoints into general toxicology studies is attractive
for several reasons, including the potential to reduce animal use and provide comprehensive
toxicity information that aids interpretation of genotoxicity results. This
laboratory has developed automated scoring techniques for monitoring two crossspecies,
blood-based genotoxicity endpoints, thereby making integration practical:
flow cytometric procedures for scoring micronucleated reticulocyte frequency and
gene mutation at the Pig-a locus. The ability to integrate these endpoints into a 28day
repeat dosing schedule was investigated with a mutagenic/non-mutagenic pair
at maximum tolerated dose levels: benzo[a]pyrene (BP, 150 mg/kg/day) and pyrene
(Pyr, 125, 250 and 500 mg/kg/day). Male Wistar Han rats were treated on Days 1-
28 via oral gavage. Blood samples obtained on Days -1, 15, 29 and 56 were analyzed
for Pig-a mutation with a dual staining method (SYTO 13 in combination
with anti-CD59-PE) that facilitated mutant cell frequency measurements in both
total erythrocytes and the reticulocyte subpopulation. Day 4 and 29 specimens
were evaluated for micronucleus frequency according to MicroFlow® Kit instructions.
BP induced robust responses on Days 15, 29 and 56 in the mutant phenotype
reticulocyte population, and on Days 29 and 56 in the mutant phenotype erythrocyte
population. No mutagenic effects in either population were apparent for
Pyr. Significant increases in micronucleated reticulocyte frequencies were observed
with BP on Days 4 and 29, whereas Pyr had no effect. These results suggest that
both endpoints are relatively specific for genotoxic activity, as each discriminated
the structurally-related mutagenic and non-mutagenic agents, even when treatment
was at a maximum tolerated dose.
1417 THE MUTAGENIC POTENTIAL OF CIS-2-BUTENE-1,
4-DIAL.
A. Terrell 1 , M. Huynh 2 and L. A. Peterson 1, 3 . 1 Environmental Health Sciences,
University of Minnesota, Minneapolis, MN, 2 Biochemistry, Molecular Biology and
Biophysics, University of Minnesota, Minneapolis, MN and 3 Masonic Cancer Center,
University of Minnesota, Minneapolis, MN.
Furan is a known rodent hepatotoxicant and carcinogen. Human exposure to furan
is widespread, but the human health effects are unknown. While there have been
many studies of furan toxicity, the mechanism of carcinogenicity is still debated.
Both genotoxic and nongenotoxic mechanisms have been proposed but in vivo and
in vitro studies have reported conflicting and inconclusive results. It is suspected
that the reactive metabolite of furan, cis-2-butene-1,4-dial, is the toxic and carcinogenic
agent. In this study, the Big Blue in vitro mutagenesis model was used to evaluate
the mutagenic potential of a single dose and chronic dose of cis-2-butene-1,4dial.
Overall, the results suggest that cis-2-butene-1,4-dial is not genotoxic in the
Big Blue in vitro mutagenesis model at the tested treatment. [funded by ES10577]
1418 THE METABOLITES OF THE AZO DYE DISPERSE RED
1 CAN REPRESENT HUMAN RISKS CONSIDERING THE
INGESTION OF CONTAMINATED WATER AND FOOD.
F. D. Chequer 1 , T. M. Lizier 2 , M. B. Zanoni 2 , R. Marcos 3 and D. P. Oliveira 1 .
1 Faculdade de Ciências Farmacêuticas de Ribeirão Preto - Universidade de São Paulo,
Ribeirão Preto, São Paulo, Brazil, 2 (2) Universidade Paulista Júlio de Mesquita
Filho, Araraquara, São Paulo, Brazil and 3 (3) Universitat Autònoma de Barcelona,
Barcelona, Spain.
Introduction: Azo dyes constitute the largest class of synthetic dyes used in commercial
applications. Following oral exposure, these dyes can be reduced to aromatic
amines by the intestinal microflora or liver azoreductases. Objectives: To determine
the products formed after oxidation and reduction of the dye Disperse Red
1, simulating hepatic biotransformation; and to evaluate the mutagenic potential of
the resultant solution. Methods: Controlled potential electrolysis was carried out
on dye solution using a Potentiostat/Galvanostat. HPLC-DAD was used for the determination
of aromatic amines generated after oxidation/reduction process. The
Salmonella/microssome assay with the strain TA98 and YG1041 with and without
S9, as well as mouse lymphoma assay (MLA) using the thymidine kinase (Tk) gene
were used for the evaluation of the mutagenicity of the solution obtained after the
reduction/oxidation process. Results: We have identified Sulfate de N-ethyl-(2-
304 SOT 2011 ANNUAL MEETING
hidroxiethyl)-p-phenyldiamine monohydrate and Nitrobenzene resultants from oxidation
and reduction of the dye Disperse Red 1. Our group showed that this azo
dye is mutagenic in different cell system. In addition after the oxidation/reduction
process, the dye still has mutagenic activity for the Salmonella/microssome assay.
We observed that the mutagenic potential of the resultant solution to the strain
YG1041 decreased after both the processes compared with the original dye, while
for the strain TA98 we observed an increase of the effect. Yet, both the original dye
Disperse Red 1 and its treated solutions showed negative results in the MLA.
Conclusions: Considering that the reduction/oxidation processes are similar to hepatic
reaction, we concluded that the ingestion of water and food contaminated
with this dye is a human health problem.
Financial support:CNPq
1419 MUTATION SPECTRUM IN HPRT-/GPT+ V79 CELLS
TREATED WITH TEAK WOOD EXTRACT AND 2-
METHYLANTHRAQUINONE, AN ABUNDANT
BIQUINONE IN TEAK WOOD.
M. J. Wilson 1 , C. Miller 1, 2 , G. Sabbioni 1, 2 and R. Rando 1 . 1 Environmental
Health Sciences, Tulane University, New Orleans, LA and 2 Tulane Cancer Center,
Tulane University, New Orleans, LA.
Wood dusts are generally classified as known human carcinogens by NTP and
IARC based on a strong association with increased risk of nasal cancer. However, no
specific carcinogenic compounds have been identified in wood dusts. Teak wood
(Tectona grandis) is an important tropical hardwood frequently used in furniture
manufacturing. An increase in mutation frequency was observed in a hprt-/gpt+
cell line following treatment with methanol extract of teak wood dust.
Fractionation of the teak dust extract using multiple analytical methods identified
2-methylanthraquinone as an abundant bioactive constituent. Large deletions of
the gpt transgene were caused by teak wood dust extract or 2-methylanthraquinone
treatments. These large signature deletions were three-fold higher in the 2-methylanthraquinone
and teak extract treatments. We conclude that teak wood dust and
its major toxic constituent, 2-methylanthraquinone, are mutagenic and may share a
common mechanism of action. 2-methylanthraquinone has not been formally
identified as a mutagen or carcinogen, but anthraquinone and other substituted derivatives
possess these activities. The presence of an abundant anthraquinone implies
teak dusts may pose an exceptional occupational risk to woodworkers.
1420 GENOTOXICITY ASSESSMENT OF
ETHYLENEDIAMINE DINITRATE (EDDN) AND
DIETHYLENETRIAMINE TRINITRATE (DETN).
G. Reddy 1 , J. Song 2 , P. Kirby 2 and M. S. Johnson 1 . 1 Directorate of Toxicology,
U.S. Army Public Health Command, Aberdeen Proving Ground, MD and 2 SITEK
Research Laboratories, Rockville, MD.
Ethylenediamine dinitrate (EDDN) and Diethylene triamine trinitrate (DETN)
are explosive compounds used in combination with other high explosives as safe,
insensitive, and inexpensive replacements. The genetic toxicity of these compounds
was evaluated to predict the potential for adverse human health effects from exposure.
The genetic toxicity test battery included: gene mutation test in bacteria
(Ames), in vitro chromosome aberration test and in vivo mouse micronucleus test.
The results of the Ames test indicated DETN was negative for Salmonella typhimurium
strains TA98, TA100, TA1535 and TA1537, and Escherichia coli strain
WP2 uvrA at doses up to 5000 μg/plate both with and without metabolic activation.
EDDN produced a positive increase in the mean number of revertants per
plate with the tester strain TA100 without activation (3.1-fold) at 5000 μg/plate.
No positive increases were observed with the other tester strains with or without activation.
The Chinese Hamster Ovary Cell (CHO) Chromosome Aberration Assay
was performed using DETN at concentrations up to 5000 μg/mL. The results indicated
that DETN did not induce structural chromosomal aberrations at concentrations
up to 5000 μg /mL in CHO cells, with or without metabolic activation.
EDDN tested up to 5000 μg/mL both without and with metabolic activation, produced
negative results in CHO cells. DETN and EDDN, when tested in vivo in
the CD-1 mouse at doses up to 2000 mg/kg, did not induce a significant increase
in the number of micronuclei in bone marrow erythrocytes. These studies demonstrate
that EDDN is mutagenic in one specific strain of Salmonella (TA100) but
were negative in other strains and in vitro for induction of chromosomal aberrations
in CHO cells and for micronuclei in the in vivo mouse micronucleus assay.
DETN was not genotoxic in both in vitro and in vivo tests. These results suggest a
low risk of genetic hazards associated with exposure to these chemicals.