Research
Reactive Nitrogen and Oxygen Species Derived
from Inflammatory Cells
Research in the Wogan
laboratory addresses two major topics,both dealing with
cancer causation, with the long-range objective of contributing
to the scientific foundation for preventive strategies.
The first focus is on how chronic inflammation acts
as a major risk factor for cancers of the stomach, colon,
liver, urinary bladder and possibly prostate and other
cancers as well. Cancers arise out of chronically inflamed
tissues, in which infiltrating macrophages and neutrophils
expose normal tissue cells to a myriad of highly reactive
chemical species that cause DNA damage, protein modification,
mutation and cell death through apoptosis and necrosis.
Our work concerns DNA damage, mutations and apoptosis
induced by nitric oxide and its reactive derivatives:
NO2, N2O3, ONOO, carbonate radical
and hydroxyl radical. These agents can either drive
cells into programmed cell death (apoptosis) through
multiple pathways, or inhibit apoptosis and enhance
mutation through damage to DNA bases, strand breaks
and cross links. Responses are highly dependent on cell
type, exposure dose and dose-rate, and underlying mechanisms
are poorly understood. We are systematically characterizing
mutagenesis, mutation spectra and chemical damage to
DNA in well-defined genetic targets exposed under controlled
conditions to nitric oxide and derivatives. Mutation
specta are compared with chemical damage induced in
the target DNA, determined through collaborations with
the Dedon and Tannenbaum groups, with the objective
of identifying DNA modifications responsible for specific
mutational events. In collaboration with the Essigmann
group, this information forms the basis for synthesis
of oligonucleotides containing appropriate modified
DNA bases, in which mutations created during replication
of the modified genomes in suitable host cells are unambiguously
identified. Mutagenesis is also studied in target cells
co-cultured with macrophages activated to produce nitric
oxide over long periods of time, as surrogates for tissue
cells exposed in vivo to prolonged inflammation.
Fluorescence Labeling of Nucleoside Adducts
for Molecular Epidemiology
The second research focus
is molecular markers of exposure to environmental carcinogens.
Accurate assessment of cancer risks from carcinogen
exposure requires quantitative data on individual human
exposure to specific carcinogens. Detection and analysis
of specific carcinogen-damaged DNA bases (DNA adducts)
resulting from endogenous or exogenous exposures to
carcinogens is essential not only for quantifying biologically
effective doses, but also for establishing relationships
between exposure and cancer risk.
To meet this need, we
have developed and validated a sensitive and specific
procedure based on fluorescence labeling of DNA adducts
combined with HPLC-laser-induced fluorescence detection.
The fluorescent dye BODIPY FL has been used to label
deoxynucleoside adducts of the aromatic amine 4-aminobiphenyl,
a bladder carcinogen widely disseminated in the environment,
and PhIP, a heterocyclic amine formed during cooking
of high-protein foods. The labeling reaction is carried
out on adducts at picomolar to nanomolar concentrations,
and the fluorescent product is detected and quantified
by HPLC/laser-induced fluorescence. This analytical
method is being used for analysis of aminobiphyenyl-deoxyguanosine
levels in DNA isolated from urinary bladder in a case-control
study of bladder cancer. The method will also be used
in evaluating the significance of oxidative DNA damage
products associated with chronic inflammation as well
as other chemical carcinogens (heterocyclic amines)
in the etiology of other major forms of cancer, including
stomach and colon. Data from these studies will be important
in developing preventive strategies to minimize the
impact of these carcinogens as risk factors.
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