Superfund Presentation***

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

2KB 1KB 0.5KB

2KB 1KB 0.5 KB

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

Acknowledgements

Duke Superfund 2015The Meyer LabDr. Joel MeyerAnthony Luz

3-photon image of DAPI stained C. elegans, Multiple-photon excitation fluorescence microscopy (University of Wisconsin-Madison, 2015)