The Toxicologist - Society of Toxicology

sue. The depth of our analysis led to the discovery of an unexpectedly large number
of hundreds of new, well-supported miRNA precursors, corresponding to almost
50% of the total number of human miRNAs deposited in the miRNA registry.
Most of the mature miRNAs encoded by these new precursors had low expression
in our samples, but can be detected in other human tissues. 49% of these new genes
were shown to be associated with Argonaute proteins, the main components of the
RNA silencing machinery, confirming that they are new functional genes.
Interestingly, more than one tenth of the new miRNAs are located in regions that
are genomically unstable in breast cancer.
The sequence data set also include non-canonical miRNAs produced by alternative
mechanisms. We found that the vault RNA (vRNA) may act as a pre-miRNA.
Vault particles are conserved organelles that have been implicated in multidrug resistance
and intracellular transport. They are formed by three different proteins and
the non-coding vault RNAs. We showed that human vRNAs are digested by Dicer
to produce several small RNAs (svRNAs). At least one of these svRNAs, we named
svRNAb, associates with Argonaute proteins to guide sequence-specific cleavage
and regulate gene expression in a manner similar to miRNAs. We also demonstrate
that svRNAb downregulates CYP3A4, a key enzyme in drug metabolism. Our
findings expand the repertoire of small regulatory RNAs and assign, for the first
time, a function to vRNAs that may help explain the observed association between
vaults and drug resistance.
900 ONCOGENIC MICRORNAS AS DRUG TARGETS FOR
CANCER CHEMOTHERAPY.
S. Safe. Veterinary Physiology, Texas A&M University, College Station, TX.
MicroRNAs (miRs) are small non-coding RNAs (18-24 nucleotides) which primarily
function to inhibit mRNA expression and it has been estimated that at least
30% of protein-encoding genes are regulated by miRs. In cancer, some miRs can
exhibit tumor suppressor- or oncogenic-like activity and therefore become potential
targets for cancer chemotherapy. Specificity protein (Sp) transcription factors Sp1,
Sp3 and Sp4 are overexpressed in cancer cells and tumors, and Sp-regulated angiogenic
(VEGF and its receptors), growth promoting (cyclin D1, c-Met and EGFR)
and survival (bcl-2 and survivin) genes are important pro-oncogenic factors. High
expression of Sp transcription factors and Sp-regulated genes has been linked to
miR-mediated inhibition of Sp repressor genes. MiR-27a is part of a miR cluster
and inhibits expression of ZBTB10, an Sp repressor, and transfection of cancer cells
with antisense-miR-27a or ZBTB10 expression plasmid decreases expression of
Sp1, Sp3, Sp4 and Sp-regulated genes. Drugs that modulate the miR-
27a:ZBTB10-Sp axis have now been identified and these agents simultaneously decrease
multiple Sp-regulated genes that are themselves individual targets for cancer
chemotherapy. Sp transcription factors and Sp-regulated genes also are repressed by
other miRs including the miR-17-92 cluster, and results of ongoing studies suggest
that overexpression of Sp genes in tumors may be due to multiple miR-Sp repressor
interactions which are also targets for anticancer drugs. Recent studies have identified
other non-coding RNAs (ncRNAs) that modulate chromatin structure and
carcinogenesis, and several agents that modify miRs also affect expression of specific
long ncRNAs. Preliminary results on drug-ncRNA interactions will be discussed.
901 MICRORNA REGULATION OF DEP-INDUCED
INFLAMMATION IN AIRWAY EPITHELIAL CELLS.
M. J. Jardim, L. Dailey and D. Diaz-Sanchez. U.S. EPA, Chapel Hill, NC.
Sponsor: T. Tal.
Morbidity and mortality attributable to air pollution continues to be a growing
problem worldwide. Despite several studies on the health effects of ambient air pollution,
underlying molecular mechanisms of susceptibility and disease remain elusive.
The epigenome controls gene expression without affecting the DNA sequence
itself and it can be inherited from generation to generation. It is not currently understood
how exposure to air pollutants can modify underlying epigenetic mechanisms
to elicit cellular responses such as inflammation. MicroRNAs are small noncoding
RNAs that have been quickly established as key regulators of gene
expression. As such, miRNAs have been found to control several cellular processes
including apoptosis, proliferation and differentiation. We have previously shown
that exposure to diesel exhaust particles (DEP) induces a rapid change in the expression
of several microRNAs in human bronchial epithelial cells (BECs).
Molecular network analysis of hsa-miR-513 suggested that the putative targets of
this miRNA were enriched for regulators of the inflammatory response, including
IL8 and COX2. We hypothesized that this miRNA plays an important role in attenuating
the inflammatory response after exposure to DEP by regulating IL8 and
COX2 mRNA levels. Indeed, over-expression of miR-513a leads to an attenuation
of DEP induced IL8 and COX2. Alternatively, induced knockdown of this
microRNA resulted in heightened levels of IL8 and COX2 mRNA in the presence
of DEP, suggesting that miR-513a may be acting as a negative regulator of the in-
flammatory response. Furthermore, induction of miR-513a upon stimulation with
DEP implies that it may play a role in a negative feedback loop which serves to attenuate
the inflammatory response once critical levels of inflammatory mediators
have been achieved. This abstract of a proposed presentation does not necessarily
reflect EPA policy.
902 IDENTIFICATION OF CHEMICAL RESPIRATORY
ALLERGENS: PRINCIPLES AND NEW
DEVELOPMENTS.
I. Kimber 1 and T. Yoshida 2 . 1 Faculty of Life Sceinces, University of Manchester,
Manchester, UK, United Kingdom and 2 Asahikawa Medical College, Asahikawa, Japan.
There are several new developments and opportunities in chemical respiratory allergy.
This is an important occupational health problem associated with significant
morbidity, and occasionally mortality. The identification and characterization of
chemical respiratory allergens has presented toxicologists with some significant
challenges, not least because there remains uncertainty about the immunological
mechanisms that may result in allergic sensitization of the respiratory tract.
Moreover, there is continuing debate about the relevant routes of exposure for the
acquisition of sensitization. Previously attention focused primarily on the development
of predictive test methods based upon animal, mainly guinea pig and mouse
models, or through exploitation of (quantitative) structure-activity relationships.
More recently, however, other strategies have been proposed and developed, included
among which are modified peptide reactivity assays, and the identification
of altered gene expression signatures specific for chemical respiratory allergens.
Recent progress will be reviewed critically and prospects for the development of
widely accepted methods for the identification and characterization of chemical respiratory
allergens will be discussed.
903 IDENTIFICATION AND CHARACTERIZATION OF
CHEMICAL RESPIRATORY ALLERGENS:CHALLENGES
AND OPPORTUNITIES.
I. Kimber. Faculty of Life Sceinces, University of Manchester, Manchester, UK,
United Kingdom.
It is now well established that a number of chemicals have the potential to induce
allergic sensitization of the respiratory tract, associated with occupational asthma.
Chemical respiratory allergy poses a number of important and intriguing challenges
to toxicologists, and there are a variety of hurdles that must be negotiated if robust
and reliable methods for hazard identification and characterization are to be developed.
In this opening presentation an historical perspective of approaches used for
the toxicological evaluation of respiratory sensitising chemicals will be provided,
with a critical appraisal of the strengths and limitations of methods that have been
proposed. In addition, the immunological mechansisms that result in the development
of allergic sensitization of the respiratory tract will be reviewed, and areas of
uncertainty highlighted and discussed. Consideration will be given also to the relevance
of IgE antibody responses for chemical respiratory allergy, and the routes of
exposure that will support the acqisition of sensitisation. Concluding comments
will focus on the opportunities that now exist for significant progress to be made.
904 ANIMAL MODELS OF CHEMICAL RESPIRATORY
ALLERGY.
J. Pauluhn. Toxicology, Bayer HealthCare, Wuppertal, Germany.
Occupational exposure priming and eliciting respiratory allergy can be attributed to
two routes: the skin and the respiratory tract. Especially in context with uncontrolled
accidental exposures, doses high enough to prime sensitization may more
readily be delivered to the skin than to the respiratory tract. Skin exposures may
lead to priming responses rendering the respiratory system more susceptible to subsequent
inhalation exposures and ensuing allergic response (asthma). This complex
inter¬relationship was investigated in a Brown Norway (BN) rat skin and inhalation
induction asthma model followed by a dose-escalation-like inhalation challenge
protocol. The latter serves the purpose to estimate the non-elicitation threshold
concentration in sensitized, asthmatic rats. Independent on the route of
induction, this was followed by three subsequent fixed concentration inhalation
challenges to the sensitizing chemical in intervals of 2 weeks. At the fourth challenge,
a dose-escalation regimen was used to determine the elicitation threshold on
‘asthmatic’ rats. Response was characterized by inflammatory endpoints in bronchoalveolar
lavage (BAL), supplemented by physiological measurements with focus
SOT 2011 ANNUAL MEETING 193