The Effect of
Dibenzo[a,l]pyrene on the
Thymus of Fetal Mice
Vivian LaRonge
Dr. William Baird’s Lab
HHMI 2005
Cancer and the Environment
Most cancers linked to the environment.
Lung and breast cancer most common for
women.
Hypotheses:
In utero exposure to DBP results in Cyp1b1mediated bioactivation and formation of DNA
adducts in the thymus.
Presence of the AhR allele plays a important role
in transplacental carcinogenesis by PAHs
PAH
Polycyclic aromatic hydrocarbons
Environmental carcinogens formed by
burning organic materials
Shape makes PAHs able to fit in DNA
DBP
Dibenzo[a,l]pyrene is the most potent PAH and is
an environmental pollutant and present in
cigarette smoke.
Fjord Region
AhR
Aryl hydrocarbon receptor carries PAHs
into the cell where they are metabolized.
Use Ah+ (“knock-in”) and Ah- (“knock-out”)
mice
Ah+ allele dominant
Knock-in mice show higher risk
Aryl hydrocarbon receptor
aromatic hydrocarbon
Cytoplasm
AhR
Protein
Translation
TNGC GTG
XRE
Nucleus
Gene
(CYP1B1)
Cyp1b1
Cytochrome P450 bioactivates many PAHs.
Cyp1b1 is specific to DBP.
Leads to tumor formation in many tissues
Cyp
Cyp
Experimental Design
AhR +/-
AhR -/For pregnant mice
15 mg DBP
gavaged gestation 17
Maternal and fetal
thymus collected
After 6h, 1d, 2d, 4d, 6d.
Isolation
Genotyping
33P
Postlabeling
Methodology
Carcinogen administered to mice by gavage 17th
day of gestation.
Maternals and fetals sacrificed; thymus tissue
removed.
Methodology cont.
Isolate DNA from fetal thymus samples.
Find DNA adducts if present, through use of 33P
Postlabeling.
Pooling of fetal thymus samples
Pooled by maternal group, genotype
2 day
6 day
M2
M1
F 1 +/-
F 2 +/-
M3
F 3 -/-
HPLC elution profiles
STANDARD
DBP
1 mV = 1000 dpm
Results
2 day – no peaks
DNA adduct peaks revealed in the 6 day fetals.
0.2119978 pmol/mg
Standard
6d-3 F 1,2 Ah +/-
Future work
Detection of DNA adducts in the thymus of
fetal and maternal mice on exposure to
DBP at 2 days.
Address the mechanisms of transplacental toxicity - Dr. Williams’ lab.
Facilitate approaches to prevention and
therapy.
Acknowledgements
Howard Hughes Medical Institute
National Institute of Health – CA90890
Dr. Kevin Ahern
Dr. William Baird
Dr. Brinda Mahadevan
Dr. David Williams
Yu Zhen
Eric Brooks
Tamara Musafia