The Toxicologist - Society of Toxicology

336 EVALUATION OF THE GENOTOXICITY AND
MUTAGENICITY OF THE DYE REACTIVE ORANGE 16.
G. R. Oliveira 1 , M. B. Zanoni 2 and D. P. Oliveira 1 . 1 Departamento de Análises
Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de
Ribeirão Preto - FCFRP/USP, Ribeirão Preto, São Paulo, Brazil and 2 Departamento
de Química Analítica, Instituto de Química - UNESP, Araraquara, São Paulo, Brazil.
Introduction: The fabrics dyeing began thousands of years ago and the commercial
availability of dyes is increasingly. In Brazil, textile industry is considered as one of
the main economic activities in the country. The dyeing process is one of the key
factors in the commercial success of textile products, since consumers are demanding
products more resistant to heat, light, perspiration and washing. This fact is relevant,
since textile dyes are discharged into the aquatic ecosystem via industrial effluents
and potentially expose humans and biota to adverse effects. The dye
Reactive Orange 16 (RO16) is widely used during the fibers dyeing process and it
has the group sulfatoethilsulfone as reactive and the group azo as cromophore.
Objective: Evaluate the mutagenicity and genotoxicity of dye RO16 using the
Salmonella mutagenicity with the strains TA98 and TA100 and comet assays in
HepG2 cells. Methodology: Comet test was carried out according to Tice et al.
(2000) with slight modifications and Salmonella mutagenicity assay was used, with
or without S9 metabolic activation, according to Maron and Ames (1983).
Different concentrations of the dye RO16 were examined for comet (1.0, 5.0, 10.0,
25.0 and 50.0 μg/mL) and for Salmonella assays (doses at a logarithmic range from
0.5 to 5000 μg/plate). Results: Dye studied induced damage in the DNA of the
HepG2 cells in a dose-dependent manner and it also showed positive mutagenic response
for TA100 strain with S9 (0.05 revertants per μg). No effect was observed
for TA98 with and without S9, as well as for TA100 in the absence of S9.
Conclusion: The use of RO16 should be avoided for dyeing textile fibers that will
be in contact with skin. Additionally, the treatment of effluent containing this dye
should be carefully evaluated in order to prevent harmful effects for human and
biota after the exposure to water and food contaminated with this compound.
337 COMPARISON OF ANEUPLOIDIES OF
CHROMOSOMES X, Y, AND 21 IN THE PERIPHERAL
BLOOD LYMPHOCYTES AND SPERM CELLS OF
WORKERS EXPOSED TO BENZENE.
Z. Ji 1 , R. H. Weldon 1 , F. Marchetti 2 , H. Chen 1 , G. Li 3 , C. Xing 2, 3 , E.
Kurtovich 1 , S. Young 1 , T. E. Schmid 2 , S. Waidyanatha 4 , S. Rappaport 1 , A. J.
Wyrobek 2 , L. Zhang 1 and B. Eskenazi 1 . 1 School of Public Health, University of
California at Berkeley, Berkeley, CA, 2 Life Sciences Division, Lawrence Berkeley
National Laboratory, Berkeley, CA, 3 China CDC, Beijing, China and 4 National
Toxicology Program, NIEHS, Research Triangle Park, NC.
Benzene, an established human carcinogen, is known to cause aneuploidy in both
human blood and sperm cells, but with considerable variation among individuals.
However, a comparison of aneuploidies induced by benzene in somatic and
germline cells within the same individuals has not been reported. We conducted a
cross-sectional study of 33 benzene-exposed male workers and 33 unexposed men
from Chinese factories to compare the frequencies of aneuploidy in their white
blood cells and sperm. Individual exposures were assessed using personal air monitoring
and urinary concentrations of benzene and trans,trans-muconic acid (E,E-
MA). Previously, we reported that benzene exposure was positively associated with
sex chromosome gains in sperm (Environ Health Perspect, 2010, 118(6):833-9). In
the present study, we examined lymphocyte aneuploidies using blood collected
within a few days of when sperm samples were provided. Stimulated peripheral
blood was cultured for 72 hrs and metaphases were examined by multicolor fluorescence
in situ hybridization with probes for chromosomes X, Y, and 21. Among
exposed workers, log-transformed urinary E,E-MA concentrations were positively
associated with sex chromosome loss in lymphocytes, in particular with loss of
chromosome X (β=0.28, p=0.01). This finding of loss of sex chromosomes in lymphocytes
contrasts with the gain of sex chromosomes in sperm from the same group
of workers. Furthermore, we found that the chromosome-specific frequencies of
aneuploidies in lymphocytes were not correlated with those in sperm. Our findings
show that occupational benzene exposure induced aneuploidies in both blood and
sperm cells, and suggest that the mechanisms of aneuploidy induction are different
between somatic and germ cells.
72 SOT 2011 ANNUAL MEETING
338 IN VITRO GENOTOXIC MECHANISMS OF ARSENIC
TRIOXIDE IN HUMAN HEPATOCELLULAR
CARCINOMA (HEPG2) CELLS: INVOLVEMENT IN
OXIDATIVE STRESS.
E. T. Brown, C. G. Yedjou and P. B. Tchounwou. Jackson State University,
Jackson, MS.
Recent studies in our laboratory indicated that oxidative stress plays a key role in arsenic
trioxide (ATO)-induced cytotoxicity in human cancer cells. In the present investigation,
we used human hepatocellular carcinoma (HepG2) cells as a model to
determine whether arsenic induced DNA damage is mediated through oxidative
stress. To achieve this goal, oxidative stress biomarkers were measured by lipid peroxidation,
glutathione peroxidase, and catalase assays, respectively. The degree of
DNA damage was estimated by the means of comet assay. The results of the lipid
peroxidation showed a significant increase (p 5-fold increase in MN was detected in tissues
treated with VB (0.0015 μg/tissue) with respect to the solvent control. Notably,
genotoxicity at this dose as measured by the standard protocol is undetectable.
Thus, the FC method appears to enhance assay sensitivity and leads to superior statistical
power, as 30x more nuclei may be scored. Although cyto B allows for the
tracking of cell division, cell cycle regulation may also be measured via propidium
iodide (PI) staining. Using this method, we detected a strong G2/M-phase arrest
induced by VB (up to 8-fold increase in the percentage of cells in G2/M) and a
moderate (< 3-fold) G2/M-phase arrest with CP at 96 h.
The results of this study indicate that the described methods simplify the evaluation
of micronuclei in the RSMN, increase its sensitivity and statistical power, and allow
for generation of valuable cell cycle information.
340 NEXT GENERATION DEL ASSAY: RAPID AND
ACCURATE ASSESSMENT OF TOXICITY.
Y. Rivina and R. H. Schiestl. Environmental Health Sciences, University of
California, Los Angeles, Los Angeles, CA.
Genetic instability and DNA deletions are involved in carcinogenesis. Furthermore,
cells from patients carrying mutations conferring cancer-prone phenotypes show a
higher level of genetic instability including DNA deletions. We have constructed
and used assays (DEL assays) that select for DNA deletion events in yeast, human
cells and in vivo in the mouse. Deletions occur by homologous recombination between
two copies of a duplicated DNA sequence to delete the intervening sequence
together with one repeat. DEL events in all three formats are inducible by a wide
variety of carcinogens including carcinogens that are negative in many other shortterm
tests. Furthermore, many cancer prone mutations highly elevated the frequency
of DNA deletions in mice adding to the solid correlation between DEL