Transcript WWW.SUNYCNSE.COM - Global Summit on Toxicology and

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
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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.
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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
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Evidence that Diet is Linked to Cancer
 Immigrants to the United States switching from a “vegetarian” diet and adopting a
meat-based 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;
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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
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Heterocyclic Aromatic Amines Are Generated
By Cooking Meats At High Temperatures:
Components:
Creatine
Sugar
Amino acids
https://encryptedtbn3.gstatic.com/images?q=tbn:ANd9GcQHO_D_eAzA_mXzZ8QGOtL9M61dTFgpmAY_u
BJxsxcsZRkKD189
http://www.slate.com/articles/health_and_science/medical_examiner/2014/01/html
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Rationale – HAAs

Heterocyclic aromatic amines (HAAs)
 Overcooked
meat - increase risk of cancer
adapted from Turesky (2004)
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AFB1 Requires Bioactivation
(CYP3A4, CYP1A1)
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Rationale – HAA activation

Metablolized by cytochrome P450
enzyme superfamily


N-acetyl transferase (NAT)
mediated acetylation


Unstable product
Nitrenium ion

adapted from Kim and
Guengerich (2005)
Ex - CYP1A2 gene
Highly reactive - forms DNA adduct
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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)
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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.
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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, 20
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Yeast AFB1 Sensitivity Requires P450 Expression
A600
1.6
BY4743 + 100 mM AFB1
1.4
1.2
BY4743 pCS316
(CYP1A2)
+ 100 mM AFB1
1
0.8
0.6
0.4
0.2
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
HRS
1.4
60 kDa
40 kDa
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 10 11 12 13 14 15 16 17
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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
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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
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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
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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
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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.
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Both CYP1A1 Alleles I462V and T461N Can
Activate AFB1 in Yeast
Recombination Frequencies
EROD Acitivities
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Transcriptional Profile of Yeast Exposed to AFB1
Other genes induced:
> 2x
TOR1
TOR2
PTK1
BIM1
MAD1
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“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
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More sensitive
More resistant
Broad Spectrum of AFB1 Resistance and Sensitivity
(Avg.) of 4300 ORFs Detected
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Genome Profiling - FunSpec
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DNA Replication Fork Integrity and DNA Damage
Tolerance Are Important In Conferring AFB1 Resistance
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
MSH6
RAD53
RAD27/FEN1
RAD30
DUN1
ESC2
UBC13
RAD17
MPH1
MRE11
RAD6
MMS2
Red color indicates meiosis specific
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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
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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
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IQ is a potent recombinagen in strains expressing
CYP1A2 and NAT2
Data was previously published in Sengstag et al. (1999)
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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
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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
500uM IQ
- 5.99 - 0% Sensitivity (+ Growth)
1.0uL DMSO - 6.67 - Control
1mM IQ
- 4.64 - 30.4% Sensitivity
rad18 expressing CYP1A2 and NAT2
Area Under Curve:
- DMSO – 7.33 – Control
- IQ
-- 5.02 – 31.5% Sensitivity
rad5 expressing CYP1A2 and NAT2
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500uM IQ
- 5.32 - 5% Sensitivity
1.0uL DMSO - 6.01 - Control
1mM IQ
- 3.58 - 40.4% Sensitivity
Expression of CYP3A4 Activates IQ
CYP1A2+NAT2
- MeOH
-- 7.64 -- Control
- 25mM IQ -- 7.55 -- 99% Growth
- 50mM IQ -- 6.17 -- 81% Growth
CYP3A4
- MeOH
-- 3.97 -- Control
- 25mM IQ -- 3.84 -- 97% Growth
- 50mM IQ -- 3.34 -- 84% Growth
- 100mM IQ -- 2.27 -- 57% Growth
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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
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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
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