The Toxicologist - Society of Toxicology

een working to define which miRNAs are testis-specific or -enriched, and to define
their changes in the blood during testicular toxicant-induced injury. This presentation
will briefly review the underlying biology and present an update of the
work in this fast-moving area.
29 Sperm mRNA Transcripts and DNA Methylation Marks As
Indicators of Testicular Injury.
K. Boekelheide. Brown University, Providence, RI.
As a pure population of cells that have matured and differentiated within the seminiferous
epithelium, sperm carry all of the information needed to fertilize an oocyte
and initiate embryogenesis. Sperm deliver small and large RNAs and DNA to the
oocyte. Using high throughput array technology, a panel of altered sperm mRNAs
has been identified after exposure of rats to testicular toxicants. In men, the extent
of sperm DNA methylation is related to sperm motility. The ultimate goal is to
identify translatable sperm molecular signatures that will allow the rapid assessment
of sperm effects in preclinical test species and exposed men.
30 Biology of Low-Dose Response for DNA-Reactive Chemicals.
J. Klapacz 1 and B. P. Engelward 2 . 1 TERC, The Dow Chemical Company, Midland,
MI; 2 Department of Biological Engineering, Massachusetts Institute of Technology,
Cambridge, MA.
In the risk assessment process, DNA-reactive agents are generally considered to
have no thresholds for their biological effects and this assumption formed the basis
for linear low-dose extrapolation of any carcinogenic effects induced by these
agents. On the other hand, cells have evolved to handle DNA lesions from endogenous
and many exogenous DNA-reactive agents. In fact, DNA-repair processes are
strictly conserved from bacteria to humans underlying their importance in protection
against the effects of these agents, such as disease and aging. In recent years,
low-dose response for DNA-reactive agents has been an active domain for research
in toxicology, with publication of several datasets with large numbers of doses focused
on the determination of no-observed-genotoxic-effect-level (NOGEL) values.
This effort has included dose-response modelling to identify the best fit between
linear and nonlinear models, such as bilinear (hockey-stick) and the
benchmark dose (BMD) suite of models, and most datasets have supported a bilinear
or nonlinear/threshold dose response as providing best fit based on statistical
criteria. However, empirical demonstration of statistically-supported
nonlinear/threshold dose response alone is not sufficient to achieve a paradigm shift
in risk assessment. A clear understanding of the biological processes behind the
shape of the low-dose response curve is similarly a critical piece of this journey, and
one where less effort has been focused to date within toxicology. This workshop will
explore some of the questions that need to be addressed to understand and bridge
DNA-repair processes and cellular responses with the mode-of-action driving these
nonlinear/threshold dose responses for genotoxic effects. The workshop will examine
existing knowledge from the field of DNA repair and link it with response to
low doses of DNA-reactive agents, in order to draw specific recommendations on a
path forward.
31 Genotoxic Effects and Dose Response: What Do We Know
So Far?
L. H. Pottenger. TERC, The Dow Chemical Company, Midland, MI.
Low-dose genotoxic response for DNA-reactive alkylating agents has been actively
investigated recently, with large datasets focused on low-dose response and determination
of no-observed-genotoxic-effect-level (NOGEL) values. Most of these recent
datasets include dose-response modelling analyses and have demonstrated a bilinear
or non-linear/threshold dose-response as the statistically supported best fit,
including datasets for directly DNA-reactive chemicals such as MMS, MNU, EMS,
and ENU. Recent data have demonstrated the ubiquitous presence of endogenously-derived
DNA adducts, some of which are identical to ones induced by exogenous
exposure to DNA-reactive chemicals. Use of stable isotopes has permitted
differentiation between the identical endogenously- and exogenously-induced
DNA adducts, demonstrating that the exogenously-induced DNA adducts can
have non-linear dose-responses at low doses; examples include formaldehyde and
EO, both of which show that endogenous adduct levels can swamp the exogenously-induced
ones at low exposures. Thus the available empirical evidence supporting
the existence of non-linear/threshold dose-response for mutagenic effects
from DNA-reactive agents is already compelling, while a growing body of evidence
6 SOT 2013 ANNUAL MEETING
is demonstrating similar non-linear/threshold dose-responses for exogenously-induced
DNA adducts. However, statistically supported demonstration of non-linear/threshold
dose-response alone is inadequate to achieve a paradigm shift in risk
assessment. A clear understanding of the biology behind the shape of the dose-response
is a critical piece for this journey, especially at low doses, although considerably
less effort has transpired on this aspect to date. Addressing the biology and biologically
plausible modes-of-action driving these non-linear/threshold
dose-responses for genotoxic effects is the next task, one in which DNA repair
mechanisms are likely to play a key role, both for adducts and for impact on doseresponse
for any resulting mutagenic effects.
32 CometChip and Recombomice Shed Light on Gene-
Exposure Interactions That Impact Genomic Stability.
B. P. Engelward. Department of Biological Engineering, Massachusetts Institute of
Technology, Cambridge, MA.
We are interested in homologous recombination (HR) and base excision repair
(BER) and the interaction between these DNA repair pathways. To explore HR in
mammals, we created mice with recombination reporter systems (Recombomice) in
which cells that have undergone an HR event fluoresce by combining tandem repeat
of truncated copies of EYFP or EGFP gene. Here, we explore genetic and environmental
factors that modulate susceptibility to HR in vivo. In particular, the
impact of inflammation, producing endogenous oxidative and alkylating DNA
damage, as both a modulator and an inducer of HR is presented. Exposure to low
dose and high dose radiation causes many of the same changes to DNA as exposure
to inflammation and can lead to severe inflammatory responses. Consequently, our
inflammation studies have broad relevance. In addition, we have developed Comet
assay called ‘CometChip’ for DNA damage analysis in single cells in an automated
fashion. The approach increases throughput and improves reproducibility. Here, we
show proof of principle of the technology as well as several applications of the
CometChip for studies of gene-environment interactions. Together, this work and
the technologies generated new biological insights.
HR has emerged as an important driver of carcinogenic sequence rearrangements.
HR repairs double strand breaks in the S/G2 phases of the cell cycle resulting from
endogenous and exogenous processes. While usually error free, errors in homologous
recombination may result in sequence rearrangements and loss of heterozygosity,
both of which are prominent features of cancer cells. BER is also a key DNA repair
pathway. In this case, damaged bases are removed, the DNA backbone is
cleaved, the ends are processed and the resulting gap is filled from the opposite
strand. Both pathways are usually error-free, but there are still rare events where
there are misalignments during HR, and misinsertions during BER. Also, both
pathways are active in response to spontaneous DNA damage, and thus would be
expected to be active in response to low dose radiation.
33 Role of Mismatch Repair in Mutagenesis, Cancer, and DNA-
Damage Signaling.
W. Edelmann. Department of Cell Biology, Albert Einstein College of Medicine,
Bronx, NY. Sponsor: J. Klapacz.
The DNA mismatch repair (MMR) system is essential for maintaining the integrity
of mammalian genomes by removing misincorporated nucleotides that result from
erroneous replication. In addition, MMR participates in the early steps of checkpoint
activation and apoptosis during the cellular response to alkylation induced
O 6 MeG:T mismatches and many other DNA lesions, particularly relevant at lowdose,
clinically-relevant exposures. Mutations in mammalian MMR genes result in
increased spontaneous mutation rates and strong predisposition to colorectal cancers
and other cancers. Eukaryotic MMR is a complex system that requires the interaction
of several MutS and MutL proteins for the initiation of the repair reaction.
Subsequent to mismatch recognition, downstream events are activated that
lead to the excision of misincorporated or damaged nucleotides and the signaling of
DNA damage-induced cell cycle arrest and apoptosis. The loss of the MMR-dependent
DNA damage response is of significant clinical relevance as it results in increased
resistance to many alkylating and chemotherapeutic agents. Our research
program focuses on elucidating the functions of the individual MutS and MutL homologs
in mammalian MMR and on assessing their importance for tumor suppression
and the DNA damage response. Our results show that the DNA damage signaling
by MMR is important for the suppression of tumorigenesis in the initial
stages of the process. In addition, we found that MMR missense mutations can effectively
separate the DNA repair and damage response functions and result in
more heterogeneous cancer phenotypes than those caused by complete loss of function
mutations.