3. Cytochrome P450 cyp1a
bioactivates 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)
4. 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
5. 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 conserved
C. elegans image under high
magnification (Utrecht University, 2015)
The mtDNA genome of C. elegans, (The
Worm Book, 2005)
6. 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)
7. Research objective
Investigate the effects of BaP exposure in
transgenic, cyp1a-expressing nematodes.
Experiments performed:
1) Growth assay
2) DNA damage assay
3) Copy number assay
8. 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
9. Dose response showing cyp1a protection from BaP exposure
Statistical significance assessed via nonparametric, Mann Whitney U test
*
*
N2 COP476 XPA-1 COP476:XPA-1
11. 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
12. 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
13. 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
14. 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
15. 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
16. 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
17. Acknowledgements
Duke Superfund 2015
The Meyer Lab
Dr. Joel Meyer
Anthony Luz
3-photon image of DAPI stained C. elegans, Multiple-photon excitation fluorescence microscopy (University of Wisconsin-Madison, 2015)
Editor's Notes
My name is Audrey Dinyari. I have been working in the Meyer lab this summer, and my research is on investigating the effects of benzo(a)pyrene exposure in transgenic, cyp1a-expressing C. elegans.
Benzo(a)pyrene, or BaP is a polycyclic aromatic hydrocarbon (PAH) composed of five benzene rings with each pair connected by two carbons. It is released into the environment from incomplete combustion of organic material such as carbon. Some sources of released BaP include wood burning, cigarette smoke and car exhaust. We wanted to look at BaP because it is a ubiquitous environmental carcinogenic toxin that causes mitochondrial as well as nuclear DNA damage.
Cytochrome P450 family of enzymes, or CYPs bioactivate BaP. This study focused on Cyp1a and how it metabolizes BaP to a DNA-reactive form. A similar enzyme, Cyp1b also bioactivates BaP. We wanted to see how expression of this gene would metabolize BaP-induced damage.
Before we can discuss the research, we need to talk about mitochondria. Mitochondria are the organelles in cells that produce energy in the form of ATP. They produce ATP via oxidative phosphorylation or OXPHOS. Each mitochondrion has its own DNA which is circular in form and exists in high copy number, or high genome copies of DNA. Mitochondrial DNA (mtDNA) are targeted by BaP due to their lack of certain repair pathways. Although mtDNA have Base excision repair pathways (BER), they lack Nucleotide excision repair pathways (NER). NER pathways repair the bulky lesions in DNA caused by damage from BaP.
In the Meyer lab, we use C. elegans as our model organism. C. elegans are microscopic nematodes found in leaf litter. They are an ideal model organism due to their simple lab maintenance, short life and reproductive cycles and availability of a broad range of genetic mutants. C. elegans’ mtDNA biology is highly conserved and has many similarities to human mtDNA. For these reasons, we study C. elegans in hopes of gaining a better insight into the health of mitochondria and how it affects the whole organism’s wellbeing. Due to the mtDNA similarities, we can use C. elegans as a model for humans in environmental health applications.
C. Elegans naturally lack the CYP1A enzyme. In order to test its expression, we used a transgenic strain of nematode that contained a zebrafish cyp1a gene. Expression and activity of CYP1A had already been confirmed from EROD assays. These assays measured the metabolism of ethoxyresorufin by CYP enzymes. The graph shows wildtype nematodes with no CYP expression, and the cyp1a-expressing nematodes with high levels of activity.
My research objective was to investigate how BaP was effecting these transgenic, cyp1a-expressing nematodes. We used three types of tests, including growth assays, DNA damage assays and DNA copy number assays.
The growth assay was conducted to observe any growth delay in BaP-exposed nematodes. Since mitochondrial function is needed for growth development, this assay would provide insight into BaP’s affect on nematode growth. The assay used four strains of nematodes. The N2 is the wildtype strain, provided as a control, XPA-1 lacked the NER function which repairs those DNA bulky lesions, COP476 expressed the cyp1a enzyme and the Cross, COP476:XPA-1 had cyp1a expression but was also NER-deficient. The nematodes were exposed in 12-well plates for 48 hours. Four different doses were used, 5, 10, 20 and 30 uM BaP doses. After 48 hours, samples were photographed and body length was measured.
So from the growth assay results we found that expression of cyp1a provided protection from BaP-induced growth delay. This shows the dose response of each strain compared over the 4 doses of BaP. The COP476 and Cross (COP476:XPA-1) strains indicated the most protection from BaP damage, with N2 following and XPA-1 with the least protection. This graph shows the data from three growth assays, collectively.
This graph shows the same data in a different configuration. Here we wanted to show the comparison between cyp1a expression and the wildtype strain (the blue and light blue columns) and also the comparison between the NER-deficient strain and the cross strain, with NER-deficiency and expression of cyp1a (orange and salmon columns) . This graph continues to show that the expression of cyp1a provides protection from BaP.
Since these results were somewhat unexpected, we decided to run PCR genotyping to confirm the strains identity. With the use of XPA-1 primers, we were able to confirm the identity of N2, and XPA-1 through gel electrophoresis. We confirmed the expression of cyp1a in the cyp1a-expressing strains using fluorescent microscopy. The insertion of cyp1a in nematodes provides a fluorescent indicator that is shown in the bottom corner. The fluorescence signifies that expression of cyp1a is present in the nematode.
Then we continued to asses DNA copy number and DNA damage in the BaP exposed nematodes. We wanted to determine if BaP was being bioactivated by the cyp1a gene. We had already observed CYP1a activity, found that CYP1A provided protection and verified the identity of the nematode strains. We wanted to look at DNA copy number it can be an indicator of DNA damage, and the copy number assay was used to normalize the damage assay. We wanted to look at DNA damage in nuclear and mitochondrial DNA because lesions in DNA may indicate damage, so this would provide more insight on how BaP damage effects the cyp1a-expressing strains.
Unexcitingly, there was not indication of significant change in copy number, when comparing BaP-dosed samples and controls. We observed mitochondrial and nuclear DNA with no significant change. We used analysis of variance to determine the significance of this data.
After testing for DNA damage, we were given more negative data. There was no significant variance in DNA damage from BaP exposure, and we once again looked at both nuclear and mitochondrial DNA. These results signify that exposure to BaP had developmental effect, but had no significant effect on DNA copy number of DNA damage.
In conclusion, this study found that presence of CYP1A did not cause BaP-induced DNA damage, BaP exposure indicated no copy number change and there was no variance between damage of nDNA and mtDNA. The cyp1a-expressing strains indicated protection from BaP damage in the growth assay. This in hypothesized to be caused by the strains safely metabolizing BaP through a different repair pathway that did not result in a DNA-reactive form, but rather a form that caused no or undetectable DNA damage.
In continuing this research, future experiments would look at the metabolism of cyp1b, which is another enzyme in the CYP family. Previous studies found approximately 40% of conserved identity of amino acids between cyp1b and cyp1a. We would be interested in cyp1b because it may form more DNA-reactive metabolites, but have overall less metabolism when compared to cyp1a.
I want to thank everyone who made this research possible and this internship a success…The Duke Superfund, the Meyer lab, the lab PI, Dr. Meyer and my mentor Anthony Luz. Thank you.
