The Effect of The Effect of Dibenzo[a,l]pyrene on the Dibenzo[a,l]pyrene on the

Thymus of Fetal Mice Thymus of Fetal Mice

Vivian LaRongeVivian LaRonge

Dr. William Baird’s LabDr. William Baird’s Lab

HHMI 2005HHMI 2005

Cancer and the EnvironmentCancer and the Environment

Most cancers linked to the environment.Most cancers linked to the environment.

Lung and breast cancer most common for Lung and breast cancer most common for women.women.

Hypotheses:Hypotheses:

In uteroIn utero exposure to DBP results in Cyp1b1- exposure to DBP results in Cyp1b1-mediated bioactivation and formation of DNA mediated bioactivation and formation of DNA adducts in the thymus.adducts in the thymus.Presence of the AhR allele plays a important Presence of the AhR allele plays a important role in transplacental carcinogenesis by PAHsrole in transplacental carcinogenesis by PAHs

PAHPAHPolycyclic aromatic hydrocarbonsPolycyclic aromatic hydrocarbons

Environmental carcinogens formed by Environmental carcinogens formed by burning organic materialsburning organic materials

Shape makes PAHs able to fit in DNAShape makes PAHs able to fit in DNA

DBPDBP

Dibenzo[a,l]pyrene is the most potent PAH and is Dibenzo[a,l]pyrene is the most potent PAH and is an environmental pollutant and present in an environmental pollutant and present in cigarette smoke.cigarette smoke.

Fjord Region

AhRAhR

Aryl hydrocarbon receptor carries PAHs Aryl hydrocarbon receptor carries PAHs into the cell where they are metabolized. into the cell where they are metabolized.

Use Ah+ (“knock-in”) and Ah- (“knock-out”) Use Ah+ (“knock-in”) and Ah- (“knock-out”) micemice

Ah+ allele dominantAh+ allele dominant

Knock-in mice show higher riskKnock-in mice show higher risk

Aryl hydrocarbon receptorAryl hydrocarbon receptor

XRE Gene (CYP1B1)

TNGC GTG

Nucleus

Cytoplasm

AhR

Protein Translation

aromatic hydrocarbon

Cyp1b1Cyp1b1

Cytochrome P450 bioactivates many PAHs.Cytochrome P450 bioactivates many PAHs.

Cyp1b1 is specific to DBP.Cyp1b1 is specific to DBP.

Leads to tumor formation in many tissuesLeads to tumor formation in many tissues

Cyp

Cyp

Experimental DesignExperimental Design

AhR -/-

For pregnant mice15 mg DBP

gavaged gestation 17

Maternal and fetal thymus collected

After 6h, 1d, 2d, 4d, 6d.

Isolation

AhR +/-

Genotyping

33P Postlabeling

MethodologyMethodology

Carcinogen administered to mice by gavage 17Carcinogen administered to mice by gavage 17 thth day of gestation.day of gestation.Maternals and fetals sacrificed; thymus tissue Maternals and fetals sacrificed; thymus tissue removed.removed.

Methodology cont.Methodology cont.

Isolate DNA from fetal thymus samples.Isolate DNA from fetal thymus samples.

Find DNA adducts if present, through use of Find DNA adducts if present, through use of 3333P P Postlabeling.Postlabeling.

Pooling of fetal thymus samplesPooling of fetal thymus samples

Pooled by maternal group, genotypePooled by maternal group, genotype

6 day

M 1 M 2 M 3

2 day

F 1 +/- F 2 +/- F 3 -/-

HPLC elution profilesHPLC elution profiles

DBPSTANDARD

1 mV = 1000 dpm

ResultsResults

2 day – no peaks2 day – no peaks

DNA adduct peaks revealed in the 6 day fetals.DNA adduct peaks revealed in the 6 day fetals.

6d-3 F 1,2 Ah +/- Standard

0.2119978 pmol/mg

Future workFuture work

Detection of DNA adducts in the thymus of Detection of DNA adducts in the thymus of fetal and maternal mice on exposure to fetal and maternal mice on exposure to DBP at 2 days.DBP at 2 days.

Address the mechanisms of trans-Address the mechanisms of trans-placental toxicity - Dr. Williams’ lab. placental toxicity - Dr. Williams’ lab.

Facilitate approaches to prevention and Facilitate approaches to prevention and therapy.therapy.

AcknowledgementsAcknowledgements

Howard Hughes Medical InstituteHoward Hughes Medical InstituteNational Institute of Health – CA90890National Institute of Health – CA90890Dr. Kevin AhernDr. Kevin AhernDr. William BairdDr. William BairdDr. Brinda MahadevanDr. Brinda MahadevanDr. David WilliamsDr. David WilliamsYu ZhenYu ZhenEric BrooksEric BrooksTamara MusafiaTamara Musafia