Determining Genetic Susceptibility to Food Carcinogens Using Saccharomyces

Determining Genetic Susceptibility to Food Carcinogens Using Saccharomyces

Determining Genetic Susceptibility to Food Carcinogens Using Saccharomyces cerevisiae (Budding Yeast). Michael Fasullo, Nick St. John, Julian Freedland, Jonathan Bard, Frank Doyle, *Patricia Egner, Thomas Begley. Colleges of Nanoscale Sciences and Engineering and Center of Excellence in Bioinformatics, State University of New York, *Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland WWW.SUNYCNSE.COM Introduction Hypothesis Hypothesis: Whereas a small fraction of cancer is caused by high penetrant genes, the majority of sporadic cancer is caused by a combination of interactions between low penetrant gene - environment interactions.

WWW.SUNYCNSE.COM Outline of talk Diet and Cancer Sources of Food Carcinogens Aflatoxin activation by CYP1A2, Heterocyclic aromatic amine activation of CYP1A2 and NAT2 Characterization of P450 polymorphisms by expression in yeast High-throughput screening for carcinogen resistance Pathways for activation of colon cancercarcinogens Conclusions and Future Directions WWW.SUNYCNSE.COM Evidence that Diet is Linked to Cancer Immigrants to the United States switching from a vegetarian diet and adopting a meatbased lower in dietary fiber have higher incidence of colorectal cancer Dietary hypotheses and diet related research in the etiology of colon cancer.

Health Serv. Rept. 88: 915 (1973) Dietary Factors and Risk of Colon Cancer in Shanghai, China Cancer Epidemiol Biomarkers Prev March 2003 12:201-208 Chinese from Qidong Province switching from a corn-based diet to a rice-based diet have a lower incidence of liver cancer Reduced Aflatoxin Exposure Presages Decline in Liver Cancer Mortality in an Endemic Region of China Cancer Prev Res October 2013 6:1038-1045; WWW.SUNYCNSE.COM WWW.SUNYCNSE.COM Aspergillus Contamination in Corn and Peanuts http://aes.missouri.edu/delta/croppest/ aflacorn.stm http://cgiarweb.s3.amazonaws.com/wp-content/uploads/2012/04/ groundnuts.jpg

AFB1 FDA limits = 20 ppb for humans, up to 500 ppb can be blended for animals WWW.SUNYCNSE.COM Heterocyclic Aromatic Amines Are Generated By Cooking Meats At High Temperatures: Components: Creatine Sugar Amino acids https://encrypted-tbn3.gstatic.com/images? q=tbn:ANd9GcQHO_D_eAzA_mXzZ8QGOtL9M61dTFgpmAY_uBJxsxcsZRkK D189 ://www.slate.com/articles/health_and_science/medical_examiner/2014/01/html

WWW.SUNYCNSE.COM Rationale HAAs Heterocyclic aromatic amines (HAAs) Overcooked meat - increase risk of cancer adapted from Turesky (2004) WWW.SUNYCNSE.COM AFB1 Requires Bioactivation (CYP3A4, CYP1A1)

WWW.SUNYCNSE.COM Rationale HAA activation Metablolized by cytochrome P450 enzyme superfamily N-acetyl transferase (NAT) mediated acetylation Unstable product

Nitrenium ion Highly reactive - forms DNA adduct WWW.SUNYCNSE.COM adapted from Kim and Guengerich (2005) Ex - CYP1A2 gene Genetic Risk Factors Involved in Food Carcinogens: Polymorphisms in P450 genes Polymorphisms in DNA repair/cell metabolism/oxidative stress response Polymorphisms in genes encoding Phase II

enzymes (NAT2, GSTa3, mouse) WWW.SUNYCNSE.COM http://ppdictionary.com/mycology/budding_yeast.jpg Why Yeast? It is a eukaryote! Budding yeast has no P450 enzyme that activates carcinogens; different genotoxic responses can be assayed after expression of different P450 enzymes 31% of yeast genes are orthologous are similar to human genes, including those that function in DNA repair, replication, and general housekeeping function

DNA damage response can be easily profiled: checkpoint activation, cell-cycle arrest, DNA adducts Systems biology approach for genetic transcription, resistance profiling, and proteomics Damage to eukaryotic organelles, such as mitochondria, can be measured. WWW.SUNYCNSE.COM Stimulation of translocations by DNA damaging agents in yeast MMS H2O2

QuickTime and a Planar RGB decompressor are needed to see this picture. MMS, rad9 H2O2, rad9 Fasullo et al., Mutat. Res 547: 123-132, 200 WWW.SUNYCNSE.COM 60 kDa 40 kDa Yeast AFB1 Sensitivity Requires P450 Expression A600

1.6 BY4743 + 100 mM AFB1 1.4 1.2 BY4743 pCS316 (CYP1A2) 1 + 100 mM AFB1 0.8 0.6 0.4 0.2 0

0 1 2 3 4 5 6 7 8 9 101112 13 1415 16 HRS 1.4 BY4743 pCS316 + 100 mM AFB1 1.2 B A600 A 58 kDa

BY4743 pCS316 1 rad52 pCS316 0.8 rad52 pCS316 + 100 mM AFB1 0.6 0.4 0.2 0 0 1 2 3 4 5 6 7 8 9 1011121314151617 WWW.SUNYCNSE.COM HRS

Cytochrome P450 1A2 alleles C406Y I386F Chevalier et al. Hum Mutat 17:355-356. http://jpet.aspetjournals.org/content/308/1/300.short Zhou et al. Mutat Res. (2004) 422 WWW.SUNYCNSE.COM Expression of P450 Polymorphisms in Yeast Rationale: Phenotype of rare polymorphisms in amino acid coding sequences are difficult ascertain by epidemiological data Full-length cDNA sequences can be expressed in yeast Biomarkers can be quantified DNA adducts Toxicity in DNA repair mutants Rad51 foci WWW.SUNYCNSE.COM

The rad4 rad51 mutant exhibits extreme AFB1 Sensitivity Black = 0 uM AFB1, blue = 25 uM AFB1, red = 50 uM AFB1 Fasullo, M. Smith, A., Egner, P and Cera, C. Activation of Aflatoxin B1 by expression of CYP1A2 polymorphisms in Saccharomyces cerevisiae,. Mutation Research 761:18-26, 2014 WWW.SUNYCNSE.COM Rad51 Foci Appear in Yeast Expressing CYP1A2 alleles Fasullo, M. Smith, A., Egner, P and Cera, C. Activation of Aflatoxin B1 by expression of CYP1A2 polymorphisms in Saccharomyces cerevisiae,. Mutation Research 761:18-26, 2014

WWW.SUNYCNSE.COM Cytochrome P450 1A1 alleles CYP1A1 alleles may be a risk factor in lung, colon, liver and breast cancer P450 Polymorphism Location Mutation Population Frequency I462V Codon 462 in

exon 7 A to G substitution 19.8% Japanese 2.2-8.9% Caucasian T461N Codon 461 in exon 7 C to A substitution 2.0-5.7% Caucasian CYP1A1

Cancer Epidemiol Biomarkers Prev. 2008 Sep;17(9):2393-401. doi: 10.1158/1055-9965.EPI-08-0326 PLoS One. 2012;7(8):e43397. doi: 10.1371/journal.pone.0043397. Cancer Lett. 2004 Aug 30;212(2):195-201. WWW.SUNYCNSE.COM Both CYP1A1 Alleles I462V and T461N Can Activate AFB1 in Yeast Recombination Frequencies EROD Acitivities WWW.SUNYCNSE.COM Transcriptional Profile of Yeast Exposed to AFB1 Other genes induced:

> 2x TOR1 TOR2 PTK1 BIM1 MAD1 WWW.SUNYCNSE.COM Humanized Yeast Deletion Collection >5,000 strains, each strain contains a deletion for a single gene Each gene deletion is marked by an antibiotic cassette and two molecular bar codes (identifiers) The presence of the strain in the pooled collection can be quantified by detecting the molecular bar codes Human CYP1A2 has been introduced into 90% of the collection WWW.SUNYCNSE.COM

More sensitive More resistant Broad Spectrum of AFB1 Resistance and Sensitivity (Avg.) of 4300 ORFs Detected WWW.SUNYCNSE.COM Genome Profiling FunSpec WWW.SUNYCNSE.COM DNA Replication Fork Integrity and DNA Damage Tolerance Are Important In Conferring AFB1 Resistan RECOMBINATIONAL REPAIR

NUCLEOTIDE EXCISION REPAIR DNA DAMAGE TOLERANCE/ REPLICATION BYPASS MISMATCH REPAIR CHECKPOINT RESPONSE MODULATION OF THE CHECKPOINT RESPONSE

DNA REPLICATION/ FORK INTEGRITY RAD51 RAD2 RAD5 MLH1 MEC1 PPH2 MUS81

RAD52 RAD14 RAD18 MSH3 TEL1 PSY2 SGS1 RAD55 RAD10

REV1 MSH4 RAD9 RTT109 RAD54 RAD1 REV3 RAD53 RAD27/FEN1 MRE11

MSH6 RAD30 DUN1 ESC2 UBC13 RAD17 MPH1 RAD6 MMS2 Red color indicates meiosis specific

WWW.SUNYCNSE.COM POL32 Summary of CYP1A2 allele expression in Yeast Full-length CYP1A2 proteins can be expressed in yeast DNA damage biomarkers can be used to differentiate AFB1 activation by polymorphic P450 enzymes Methodology could be expanded to other CYP1A2 alleles or genotoxins WWW.SUNYCNSE.COM Mechanisms of Activation of IQ By Phase I and II Enzymes IQ activation by CYP1A2 and NAT2

IQ Glucuronidation and Excretion In the Bile IQ reactivation by Microbial Glucoronidases in the Colon or IQ activation by colon-specific CYPs WWW.SUNYCNSE.COM IQ is a potent recombinagen in strains expressing CYP1A2 and NAT2 Data was previously published in Sengstag et al. (1999) WWW.SUNYCNSE.COM

Genome Profiling Expression of NAT2 Enhances Genotoxicity rad4 rad51 expressing CYP1A2 CYP1A2 - 3.6 mM IQ -- 11% Sensitivity - 3.2 mM IQ -- 1% Sensitivity rad4 rad51 expressing CYP1A2 and NAT2 CYP1A2+NAT2 - 3.6 mM IQ -- 50% Sensitivity - 2.7 mM IQ -- 26% Sensitivity - 1.5 mM IQ -- 1% Sensitivity WWW.SUNYCNSE.COM DNA damage tolerance mutants expressing

CYP1A2 and NAT2 are also IQ sensitive rad18 expressing CYP1A2 Area Under Curve: - DMSO 6.98 Control - IQ -- 5.35 23.4% Sensitivity rad5 expressing CYP1A2 rad18 expressing CYP1A2 and NAT2 Area Under Curve: - DMSO 7.33 Control - IQ -- 5.02 31.5% Sensitivity rad5 expressing CYP1A2 and NAT2

500uM IQ - 5.99 - 0% Sensitivity (+ Growth) 500uM IQ - 5.32 - 5% Sensitivity 1.0uL DMSO - 6.67 - Control 1.0uL DMSO - 6.01 - Control WWW.SUNYCNSE.COM 1mM IQ - 4.64 - 30.4% Sensitivity 1mM IQ - 3.58 - 40.4% Sensitivity Expression of CYP3A4 Activates IQ CYP3A4 CYP1A2+NAT2 - MeOH

-- 3.97 -- Control - MeOH -- 7.64 -- Control - 25mM IQ -- 3.84 -- 97% - 25mM IQ -- 7.55 -- 99% Growth Growth - 50mM IQ -- 3.34 -- 84% - 50mM IQ -- 6.17 -- 81% WWW.SUNYCNSE.COM Growth Growth Conclusions AFB1 is a strong genotoxin in yeast, biomarkers include DNA adducts and Rad51 foci.

Both CYP1A2 and CYP1A1 polymorphisms can be expressed and phenotyped in yeast Heterocyclic aromatic amines, such as IQ, are recombinagens in yeast and DNA repair mutants are IQ sensitive Yeast Libraries are useful in identifying AFB1-sensitive genes and for screening other CYP-activated xenobiotics and drugs Future Directions: Determine whether orthologous genes that confer resistance to AFB1 also do so in mice

WWW.SUNYCNSE.COM Acknowledgements and Collaborators Mingzeng Sun (postdoctoral fellow) and William Bortcosh (Albany Medical School) Monica Keller-Seitz and Christian Sengstag (Swiss Insitutte of Technology) Chris Vulpe (UC Berkeley) NIH Funding, ES021133 WWW.SUNYCNSE.COM

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