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Investigating the effects of Benzo(a)pyrene exposure in transgenic, cyp1a-expressing Caenorhabditis elegansAudrey DinyariMeyer LabJuly 22nd 2015

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.1

Benzo(a)pyrene (BaP)Polycyclic aromatic hydrocarbon (PAH)Generated by incomplete combustion Wood burningCigarette smokeCar exhaustUbiquitous environmental carcinogenic contaminantCauses nDNA damage and mtDNA damage

Benzo(a)pyrene, ( NCBI, 2015)

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.2

Cytochrome P450 cyp1abioactivates BaPCYPs metabolize BaPCyp1a can bioactivate BaP to a DNA-reactive formCyp1b 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)

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.3

MitochondriaProduce ATP via Oxidative phosphorylation Contain own circular genomes (mtDNA) exist in high copy number in cells (100s 10,000s)BaP preferentially targets mtDNAMitochondria lack NER repair pathwayRepairs bulky BaP-induced DNA damage

Villarreal, 2006Kalicharan, 2008

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.

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Why Caenorhabditis elegans?C. elegans are microscopic nematodes that live in leaf litterIdeal lab model due to: Simple maintenanceShort life cycle & reproductive cycle Availability of genetic mutantsMitochondrial biology and genome is conserved

C. elegans image under high magnification (Utrecht University, 2015)

The mtDNA genome of C. elegans, (The Worm Book, 2005)

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 organisms wellbeing. Due to the mtDNA similarities, we can use C. elegans as a model for humans in environmental health applications.5

C. elegans lack CYP1AC. elegans lack CYP1 family of enzymesTransgenic strain expresses zebrafish cyp1aEROD 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)

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.6

Research objectiveInvestigate the effects of BaP exposure in transgenic, cyp1a-expressing nematodes.

Experiments performed:1) Growth assay2) DNA damage assay3) Copy number assay

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.7

Growth Assay SetupMitochondrial function is required for larval development4 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 BaPMeasured body length

12-well plate, used in growth assaysCon1% DMSO5uM10uM20uM30uM

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 BaPs 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.8

Dose response showing cyp1a protection from BaP exposure

Statistical significance assessed via nonparametric, Mann Whitney U test**N2COP476XPA-1COP476:XPA-1

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.9

********Statistical significance assessed via nonparametric, Mann Whitney U test

********cyp1a protects larval nematodes as they develop from BaP exposure

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.10

Genotyping and Strain ConfirmationUsed PCR to confirm identity of strains Xpa-1 primers confirmed strain identityConfirmed identity of cyp1a strain via fluorescent indicator (below)

Photo credit, Latasha Smith

2KB1KB0.5KB2KB1KB0.5 KB

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.11

Why look at copy number and DNA damage?Determine if BaP is being bioactivatedObserved CYP1A activity (EROD assay)Established CYP1A provides protection in developing nematodesVerified 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 & nDNAIndicates DNA damageUse to normalize DNA damage assayDNA damage assay:Increase in lesions may indicate increased damageLesions measured per 10KB of DNA

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.12

BaP exposure did not affect mtDNA or nDNA copy number

Nuclear DNA copy numberMitochondrial DNA copy number2-way ANOVA, P=0.28 mtDNA, 0.28 nDNA Analysis of variance (ANOVA) used to assess DNA damage and copy number values COP476:XPA-1XPA-1COP476:XPA-1XPA-1

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.

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DNA damage was not detected in mtDNA or nDNA after BaP Exposure

Mitochondrial DNA damageNuclear DNA damage2-way ANOVA, P=0.31 mtDNA, 0.56 nDNA COP476:XPA-1XPA-1COP476:XPA-1XPA-1

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.14

ConclusionsPresence of CYP1A did not result in BaP-induced DNA damageNo indicated change in copy number or variance between nDNA and mtDNA damage Cyp1a-expressing strains were protected from growth delay, induced by BaP exposureHypothesis- 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

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.15

Future DirectionsFurther experiments would include the role of cyp1b and the metabolism of BaPPrevious 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

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.16

AcknowledgementsDuke 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)

I want to thank everyone who made this research possible and this internship a successThe Duke Superfund, the Meyer lab, the lab PI, Dr. Meyer and my mentor Anthony Luz. Thank you.

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