NEW METHODOLOGY FOR PHENOTYPING P450 POLYMORPHISMS AND SUSCEPTIBILITY

NEW METHODOLOGY FOR PHENOTYPING P450 POLYMORPHISMS AND SUSCEPTIBILITY

NEW METHODOLOGY FOR PHENOTYPING P450 POLYMORPHISMS AND SUSCEPTIBILITY TO ENVIRONMENTAL TOXICANTS USING HUMANIZED BUDDING YEAST Michael Fasullo, Cinzia Cera, Jonathan Bard, Frank Doyle, Julian Freedlander, *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 Preliminary and CNSE Confidential cnse.albany.edu Outline of talk

Aflatoxins and hepatocarcinogens Activation of aflatoxins Biomarkers for aflatoxin exposure in yeast Characterization of P450 Polymorphisms High-throughput screening for AFB1 resistance Identity of Candidate Genes Conclusions and Future Directions 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

Hepatocellular Carcinoma is Concentrated in AFB1 Contaminated Areas in China Hepatocellular carcinoma incidence correlates with aflatoxin B1 (AFB1) and HBV exposure Specific P53 S(249) mutation is found in HCC corresponding to AFB1 exposure Sell, Cancer Res. 63:7554, 2003 WWW.SUNYCNSE.COM AFB1 Requires Bioactivation (CYP3A4) WWW.SUNYCNSE.COM Risk Factors Involved in

Hepatocarcinogenesis: Aflatoxin exposure Hepatitis B exposure Detoxification by Phase II enzymes (GSTa3, mouse) Polymorphisms in P450 genes Polymorphisms in DNA repair/cell metabolism/oxidative stress response 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 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 The rad4 rad51 mutant exhibits extreme AFB1 Sensitivity Black = 0 uM AFB1, blue = 25 uM AFB1, red = 50 uM AFB1 WWW.SUNYCNSE.COM

Transcriptional Profile of Yeast Exposed to AFB1 Other genes induced: > 2x TOR1 TOR2 PTK1 BIM1 MAD1 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 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 Coumassie Stain Western Concentrations of AFB1 7N-Gua Adducts in DNA. a Indicates CYP1A2 allele expressed in yeast b

N > 2, generated after exposure to 50 mM AFB1 Adducts CYP1A2 allele/Adducts WT, numbers in bold indicate significant differences with wild type. c 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 AFB1 Toxicity in rad4 rad51 mutants expressing P450 Polymorphisms 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 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 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 Genome Profiling Multiplex Primers Centralized KanMX4 gene Flanked by uptag and dntag (~20-25 unique BP) Bound 4-nt multiplex index and the Illumina sequencing primer sequence WWW.SUNYCNSE.COM

Genome Profiling - Analysis Uptags counted for three different exposures Normalize counts/Binomial Statistics Compare log2N ratios (treated/untreated) For three independent experiments Collect sensitive and resistant strains Group into Gene Ontology groups

Validate groups by survival curves 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 Representative deletions strains that exhibit AFB1 sensitivity A600

1.5 BY4743 1 1.4 0 mM AFB1 10 mM AFB1 100 mM AFB1 1.2 rad18 1 50 mM AFB1 0.8

0.5 0 mM AFB1 10 mM AFB1 100 mM AFB1 0.6 0.4 0 0.2 0 HRS A600 1.5 mms2

1 0.5 0 HRS 0 mM AFB1 10 mM AFB1 100 mM AFB1 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 WWW.SUNYCNSE.COM

HRS ftr1 HRS 0 mM AFB1 10 mM AFB1 100 mM AFB1 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 Conclusions AFB1 is a strong genotoxin in yeast, biomarkers include DNA adducts and Rad51 foci. Resistance genes includes DNA damage tolerance and downregulation of the Checkpoint Response Libraries may be useful 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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