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Investigating the effects of Benzo(a)pyrene exposure in transgenic,
cyp1a-expressing Caenorhabditis elegans
Audrey DinyariMeyer Lab
July 22nd 2015
Benzo(a)pyrene (BaP)• Polycyclic aromatic
hydrocarbon (PAH)• Generated by incomplete
combustion – Wood burning– Cigarette smoke– Car exhaust
• Ubiquitous environmental carcinogenic contaminant
• Causes nDNA damage and mtDNA damage
Benzo(a)pyrene, ( NCBI, 2015)
Cytochrome P450 cyp1abioactivates BaP
• CYPs metabolize BaP• Cyp1a can bioactivate
BaP to a DNA-reactive form– Cyp1b is also capable of
bioactivating BaP
Cytochrome P450, family 1, subfamily A, polypeptide 2 (Protein Data Bank)
Major metabolic pathways and formation of DNA adducts of BaP (Arlt et al. 2014)
Mitochondria• Produce ATP via
Oxidative phosphorylation
• Contain own circular genomes (mtDNA) – exist in high copy number
in cells (100s – 10,000s)• BaP preferentially targets
mtDNA• Mitochondria lack NER
repair pathway– Repairs bulky BaP-
induced DNA damage
Villarreal, 2006
Kalicharan, 2008
Why Caenorhabditis elegans?
• C. elegans are microscopic nematodes that live in leaf litter
• Ideal lab model due to: – Simple maintenance– Short life cycle &
reproductive cycle – Availability of genetic
mutants• Mitochondrial biology and
genome is conservedC. elegans image under high magnification (Utrecht University, 2015)
The mtDNA genome of C. elegans, (The Worm Book, 2005)
C. elegans lack CYP1A• C. elegans lack CYP1
family of enzymes• Transgenic strain
expresses zebrafish cyp1a
• EROD Assay- confirmed CYP1A activity
EROD assay showing enzyme activity in nematodes and killifish (The Meyer Lab)
Metabolism of ethoxyresorufin by CYP enzyme (USGS Columbia Environmental Research Center, 2015)
Research objective
Investigate the effects of BaP exposure in transgenic, cyp1a-expressing nematodes.
Experiments performed:1) Growth assay2) DNA damage assay3) Copy number assay
Growth Assay Setup• Mitochondrial function is
required for larval development• 4 strains of nematodes used:– N2 (Wild-type)– XPA-1 (NER-deficient)– COP476 (CYP1A expressing)– COP476:XPA-1 (CYP1A expressing
& NER-deficient)
• 48 hour exposure • 5, 10, 20 and 30 uM BaP• Measured body length
12-well plate, used in growth assays
Con 1% DMSO 5uM 10uM
20uM 30uM
Dose response showing cyp1a protection from BaP exposure
Statistical significance assessed via nonparametric, Mann Whitney U test
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N2 COP476 XPA-1 COP476:XPA-1
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Statistical significance assessed via nonparametric, Mann Whitney U test
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cyp1a protects larval nematodes as they develop from BaP exposure
Genotyping and Strain Confirmation• Used PCR to confirm
identity of strains • Xpa-1 primers
confirmed strain identity
• Confirmed identity of cyp1a strain via fluorescent indicator (below)
Photo credit, Latasha Smith
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Why look at copy number and DNA damage?
Determine if BaP is being bioactivated Observed CYP1A activity (EROD assay) Established CYP1A provides protection in developing nematodes Verified strains via PCR
Since CYP1A bioactivates BaP, we wanted to investigate DNA damage and copy number in these strains.
DNA copy number assay:– Genome copies in mtDNA & nDNA– Indicates DNA damage– Use to normalize DNA damage assay
DNA damage assay:– Increase in lesions may indicate increased damage– Lesions measured per 10KB of DNA
BaP exposure did not affect mtDNA or nDNA copy number
Nuclear DNA copy number
Mitochondrial DNA copy number
2-way ANOVA, P=0.28 mtDNA, 0.28 nDNA
Analysis of variance (ANOVA) used to assess DNA damage and copy number values
COP476:XPA-1 XPA-1
COP476:XPA-1 XPA-1
DNA damage was not detected in mtDNA or nDNA after BaP Exposure
Mitochondrial DNA damage
Nuclear DNA damage
2-way ANOVA, P=0.31 mtDNA, 0.56 nDNA
COP476:XPA-1 XPA-1
COP476:XPA-1 XPA-1
Conclusions
• Presence of CYP1A did not result in BaP-induced DNA damage
• No indicated change in copy number or variance between nDNA and mtDNA damage
• Cyp1a-expressing strains were protected from growth delay, induced by BaP exposure
• Hypothesis- cyp1a-expressing strains were able to safely metabolize BaP through a pathway that did not result in the bioactivation of BaP, thus causing no or undetectable DNA damage
Future Directions
• Further experiments would include the role of cyp1b and the metabolism of BaP
• Previous studies have found ~40% conserved identity of amino acids of cyp1b with cyp1a (Lee et al. 2003)
• CYP1B- May form more DNA-reactive metabolites, but has less overall metabolism compared to CYP1A