The Toxicologist - Society of Toxicology

118 TO STUDY THE SIGNAL TRANSDUCTION ON 2-ABP &
4-ABP INDUCED DNA DAMAGE IN HEP G2 CELL.
J. Wong 1 , J. Wu 2 , S. Chen 1 and L. Chen 3 . 1 Department of Biotechnology, National
Kaohsiung Normal University, Kaohsiung, Taiwan, 2 Department of Applied
Chemistry, Fooyin University, Kaohsiung, Taiwan and 3 Department of Medical
Nutrition, I-Shou University, Kaohsiung, Taiwan.
The International Agency for Research on Cancer declared that 4-aminobiphenyl
(4-ABP) was carcinogenic to human. 2-aminobiphenyl (2-ABP) is its analogue.
Our previous results showed that 4-ABP and 2-ABP were examined for their ability
to induce oxidative DNA damage in Hep G2 cells. However, the molecular and cellular
mechanisms underlying 2-ABP & 4-ABP associated genotoxicity are not fully
understood. Our goal in this study was to determine the DNA damage signaling
pathway. We found that 2-ABP and 4-ABP could induce γ-H2AX phosphorylation,
a sensitive DNA damage marker, in Hep G2 cells, suggesting that DNA damages
were elicited by 2-ABP and 4-ABP. Further, Hep G2 cell were incubated with
200 μM2-ABPand200μM 4-ABP for various periods, that induced the activation
of mitogen-activated protein kinase family (MAPK) proteins including JNK and
ERK 1/2 were detected in 1h. In addition, we found 2-ABP treatment inhibited
cell growth by inducing G0/G1 phase arrest. Whereas we suggest that 2-ABP and
4-ABP could induce DNA damage and activation MAPK phosphorylation but 2-
ABP can only induced cell cycle arrest. Our findings suggested that the mechanisms
of induced DNA damage in Hep G2 cell by 2-ABP and 4-ABP were different.
119 GENOTOXICITY AND CHANGES IN GENE
EXPRESSION PROFILES INDUCED BY AIRBORNE
PARTICLES IN HUMAN CELL LINES.
J. Topinka, K. Uhlirova, H. Libalova, A. Milcova, J. Schmuczerova and R. J.
Sram. Genetic Ecotoxicology, Institute of Experimental Medicine AS CR, Prague 4,
Czech Republic.
Human embryonic lung fibroblasts (HEL) and human alveolar adenocarcinoma
cells A549 (model of human lung epithelial cells) were employed to assess the genotoxicity
induced by complex mixtures of organic air pollutants adsorbed onto respirable
air particles (PM2.5) collected by high volume samplers in 4 localities of the
Czech Republic (Ostrava-Bartovice, Ostrava- Poruba, Karvina a Trebon) differing
by the extent of the air pollution. To get more insight into the mechanisms of the
toxic effects, changes in gene expression profiles caused by organic extracts from
PM2.5 particles in HEL and A549 cells were investigated. For this purpose, DNA
adduct forming activity of extractable organic matter (EOM) from the PM2.5 particles
was analyzed by 32P-postlabelling at subtoxic EOM concentrations of 10, 30,
and 60 ug/ml. Simultaneously, whole genome expression profiles using Illumina
reader and Human HT-12 v2 Expression BeadChip Kit were determined. For both
HEL and A549 cells, dose dependent increase of DNA adduct levels was found.
Comparable adduct levels were induced by various EOMs in both cell types. Close
correlation between DNA adduct levels induced by all EOMs in both cell lines and
c-PAHs concentrations in EOMs suggests that genotoxicity of the polluted air is
much closely related to c-PAHs content than to PM2.5. The results further suggest
that ferrous metallurgy and coke oven emissions in the proximity of Ostrava-
Bartovice are much stronger sources of genotoxic compounds forming DNA
adducts than traffic emissions in Ostrava-Poruba (3-fold higher traffic density).
First analysis of gene expression data suggests that multiple genes were deregulated
by EOM by dose-dependent manner. Biological processes (metabolic and signaling
pathways) affected by organic air pollutants bound on PM2.5 were identified in
Ostrava-Bartovice (polluted mostly by industrial emissions) and in Ostrava-Poruba
(mostly traffic related air pollution). Supported by the Czech Ministry of
Education (grant #2B08005) and by AS CR (grant AV0Z50390512).
120 MOLECULAR DETERMINANTS OF DNA POLYMERASE
ETA FIDELITY.
S. C. Suarez and S. D. McCulloch. Environmental and Molecular Toxicology, North
Carolina State University, Raleigh, NC.
Y-family DNA polymerases are required to bypass lesions encountered during
DNA replication. This bypass prevents replication fork collapse and DNA strand
breaks. UV light can cause not only cyclobutane pyrimidine dimers (CPD), but
also oxidative damage such as 7,8-dihydro-8-oxo-guanine (8-oxo-G). 8-oxo-G is a
lesion that is generated thousands of times per day in each human cell under normal
conditions as a consequence of cellular respiration. DNA polymerase eta (pol
eta) is able to bypass a number of lesions that can block replication fork progression,
including CPD and 8-oxo-G. Recent reports of pol eta crystal structures complexed
with CPD show a more open active site, as well as implicating a number of
active site residues that play a role in the bypass of CPD. To investigate the role of
these residues in the bypass of 8-oxo-G, we have created and purified a set of single
amino acid substitution mutants of pol eta. We used these mutants to test the effect
of these residue changes on ability of the polymerase to bypass the lesion at template
to enzyme ratios and time points that approximate a lesion bypass event in
vivo. We also used an assay to examine the effect of these mutants on the insertion
fidelity of pol eta past 8-oxo-G. Our results indicate that these residue changes can
alter both the fidelity and bypass of the enzyme. Some changes can confer a mutator
phenotype, while others have an opposite effect. These changes can also reduce
the ability of the polymerase to bypass 8-oxo-G. These results indicate that pol eta
walks a fine line between the ability to bypass lesions while still maintaining adequate
replication fidelity. The increase in bypass at the cost of decreased fidelity has
implications for an organism at multiple levels. The characterization of these mutants
gives us insight as to the molecular determinants for the most important properties
of pol eta in performing its function of bypassing lesions and preventing mutation
during lesion bypass.
121 MECHANISMS OF THE WERNER SYNDROME
PROTEIN IN PROTECTING AGAINST CR(VI)
INDUCED TELOMERE LOSS.
F. Liu, K. E. Knickelbein, A. Barchowsky and P. L. Opresko. Environmental and
Occupational Health, University of Pittsburgh, Pittsburgh, PA.
Werner syndrome protein (WRN) is a DNA helicase-exonuclease that facilitates
telomeric DNA replication. Telomeres consist of guanine-rich DNA repeats coated
by specialized proteins that protect chromosomal ends from being processed as
DNA breaks. Disruption of telomere replication can lead to dysfunctional telomeres,
which contribute to aging related diseases and cancer. However, limited information
is available regarding the effects of environmental exposures on telomere
stability. Hexavalent chromium (Cr(VI)) is an established environmental risk factor
for lung cancer in the occupational setting. We showed previously that WRN suppresses
Cr(VI)-induced telomere loss and are currently investigating the mechanism.
Since Cr(VI) induces adducts that arrest DNA polymerases predominantly in
contiguous guanine runs, we predict that these lesions block telomere replication.
We found Cr(VI) caused oxidative stress only at high levels (>100 μM), eliminating
this as a mechanism for telomere loss. Cr(VI) increased foci formation of polymerase
eta, which synthesizes past DNA lesions that block replicative polymerases.
Thus, Cr(VI)-induced polymerase eta localization at telomeres serves as a marker
for telomeric replication blocking lesions. We found WRN deficiency resulted in
delayed reinitiation of Cr(VI)-induced stalled replication forks. We are using chromosome-orientation
fluorescent in situ hybridization to examine WRN roles in repairing
Cr(VI)-disrupted replication forks at telomeres by homologous recombination.
Furthermore, we are testing WRN roles in suppressing Cr(VI)-induced DNA
deletions and mutagenesis in telomeric DNA using a shuttle vector system harboring
the supF gene and telomere repeats as a mutagenic target. Our study indicates
that Cr(VI) generated DNA replication but not oxidative stress contributes to
telomere loss at occupationally relevant levels and that WRN facilitates repair of
Cr(VI)-disrupted telomere replication.
122 ANALYSIS OF MOLECULAR SPLINT MUTANTS OF
HUMAN DNA POL η AND THEIR EFFECT ON
POLYMERASE PROPERTIES.
R. A. Beardslee and S. D. McCulloch. Department of Environmental and Molecular
Toxicology, North Carolina State University, Raleigh, NC.
DNA polymerase η (Pol η) is responsible for the bypass of cyclobutane pyrimidine
dimers (CPDs) during DNA replication as well as other lesions such as 8-oxoguanine
(oxoG). Both are ubiquitous lesions; the former is produced by exposure to
UV radiation, while the latter is generated by reactive oxygen species (ROS) yielded
during xenobiotic metabolism in addition to normal cellular function. It follows
that Pol η is indispensable to successful completion of DNA replication and organism
survival. It has been recently reported that a β-strand (amino acids 316-324) in
the little finger region of Pol η appears important to correctly align the template
strand with the catalytic core of the enzyme. We hypothesized that modification of
these residues would interfere with correct enzyme-DNA alignment and alter Pol η
activity and fidelity. To study the role of these mutations, we expressed catalytic
core amino acids 1-511 of human Pol η in E. coli. We generated both wild type enzyme
as well as enzyme with single amino acid substitutions at residues Pro-316,
Thr-318, Gly-320, Ser-322 and Asn-324. Overexpressed protein was purified by
chromatography using HiTrap Chelating HP (GE) with subsequent application
of Pol η rich fractions to Mono S (GE). Purified protein fractions and DNA
SOT 2011 ANNUAL MEETING 25