Transplacental DNA Adduct Formation by
Dibenzo[a,l]pyrene in Mice
Daniel J. Albershardt
Senior, Dept. of Biochemistry/Biophysics
Dr. William M. Baird Laboratory
Overview
• Introduction
• Methods
• Summary
Polycyclic Aromatic Hydrocarbons (PAH)
• Found in complex environmental pollutants
• Formed by incomplete combustion of organic compounds
• Human exposure linked to cancer incidence
Dibenzo[a,l]pyrene (DBP)
Dibenzo[a,l]pyrene
• Related compounds have been shown to be
transplacental
Anderson, LM, Ruskie, S, Carter, J, Pittinger, S, Kovatch, RM, Riggs, CW.
Pharmacogenetics (1995) 6:364-72
PAH
Metabolism
• Many sites can be epoxidated.
• Epoxides are unstable.
• Epoxide hydrolase may hydrolyze
the epoxide to a diol.
• Subsequent epoxide is a more
stable, reactive electrophile.
Chen J, et. al. "MMDB: Entrez's 3D-structure database", Nucleic Acids Res. 2003 Jan; 31(1): 474-7.
Mutations by Dibenzo[a,l]pyrene
(-)-anti-DB[a,l]PDE [nM]
Types of Sequence
Alteration
DMSO
1
2
5
10
Base Substitutions
8(73%)
11(79%)
16(67%)
25(74%)
38(81%)
Exon Deletions
3(27%)
3(21%)
3(12%)
3(9%)
1(2%)
±1 Frameshifts
0
0
4(17%)
2(6%)
4(9%)
Tandem Mutations
0
0
1(3%)
2(6%)
1(2%)
Small Insertions
0
0
0
1(3%)
1(2%)
Total
11(100%)
14(100%)
24(100%)
33(100%)
45(100%)
No. of Mutant
Clones Analyzed
7
14
14
14
15
B. Mahadevan, W.-M. Dashwood, A. Luch, A. Pecaj, J. Doehmer, A. Seidel, C. Pereira, and W. M. Baird.
Environ. Mol. Mut. 41:131-9 (2003)
Objectives
• Determine transplacental DNA adduct
formation by DBP in mice
• Relevance to human health
Methods
17th Day Gestation
15mg DBP/kg body mass
Sacrifice via CO2 (maternal)
or decapitation (fetal)
Lung and liver removed
DNA isolated and 33P postlabeled
HPLC Analysis
HPLC Profiles
Controls:
DBP
STANDARD
Maternal Lung Tissue HPLC Profiles
6 Hours
2 Days
1 Day
4 Days
6 Days
Total DNA Adducts in
Maternal Lung Tissues
Total DNA Adducts (pmol/mg)
30
25
20
15
10
5
0
6 hours
1 Day
2 Days
4 Days
6 Days
Fetal Lung Tissue HPLC Profiles
2 Day Exposure
Total DNA Adducts After 2 Days Exposure
30
Total DNA Adducts (pmol/mg)
25
20
15
10
5
0
Maternal
P1
P2
P3
P4
P5
P6
P7
Fetal Liver Tissues
• No DNA adducts detected using
our method
Summary
• DNA adducts found in maternal lung tissues at
all exposure times
• 2 days exposure to DBP formed DNA adducts in
fetal lung tissue
• No DNA adducts detected in fetal liver tissue
after 2 days exposure
Future Research
• DNA adduct determination of:
Maternal Liver
Fetal Lung and Liver at Different
Exposure Times
Acknowledgements
• Dr. William M. Baird
• Dr. Kevin Ahern
• Dr. Brinda Mahadevan
• Tamara Musafia
• Dr. David Williams
• Yu Zhen
• Mandy Louderback
• Howard Hughes Medical
Institute
• LARC