Acknowledgements This research was funded by the California Breast Cancer Research Program (#14IB-0032) and the Susan G. Komen for the Cure (#DISPO707183) to P.K.M. The

study was possible with the collaboration of the United Farm Workers, and Ms. Jennifer Dodge and Kristine Ongaigui at UCSF-Fresno.

Yesenia Ibarra*, Kathryn Patterson*, Malika Sahni*, Jessica Borba*, Jason A. Bush*†, and Paul K. Mills†

*Department of Biology, California State University, Fresno †Department of Internal Medicine, UCSF-Fresno

Objective

The long-term goal of this study is to determine the association between exposure to pesticides and

risk of hormone-dependent cancer (breast or prostate) in the Hispanic population of the intensely

agricultural San Joaquin Valley of California.

BxPc-3

P4.03 Methodology

.

Discussion & Future Directions GSTM1 null – No significant correlation with breast cancer risk (O.R.=0.99, 95% CI=0.28-3.51) unless associated with Endosulphan exposure.

GSTT1 null – Breast cancer risk doubled (O.R.=2.21, 95% CI=0.39-12.63)

GSTP1 (I105V) & (A114V) – Elevated risk of breast cancer in the heterozygous or homozygous variant allele.

CYP1B1 (A119S) – No significant association with breast cancer risk (O.R.=0.77, 95% CI=0.14-3.70)

(V432L) – Elevated risk of breast cancer (O.R.=2.33, 95% CI=0.64-8.54) suggesting that women carrying the homozygous Val allele had higher risk

than those women with the Leu/Leu genotype.

Prostate cancer risk will be analyzed similarly to the breast cancer study for comparative analysis of GSTM1, GSTT1, CYP1A1, and CYP1B1. In addition, we

will investigate methylation status to examine epigenetic changes using the PyroMarkQ24 pyrosequencing instrument at the Fresno State RIMI Functional

Genomics & Proteomics Facility.

Although this study is of low volume of participants, it indicates that it is feasible to identify, trace, consent and recruit Hispanics in the San Joaquin Valley of

California who have recently been diagnosed with breast cancer or prostate cancer.

Background In California alone, there will be an estimated 25,510 new cases of breast cancer and 25,030 new

cases of prostate cancer diagnosed (American Cancer Society, 2011).

California leads the nation in agricultural production and uses over 170 million pounds of active pesticide ingredients each year (California Dept of Pesticide Regulation, 2007).

• 42 Female Hispanic participants

• DNA extractions from Oragene kits1

• PCR using annealing primers2

• PCR product goes to 1 of 3 paths

• (A) Sequencing3

• (B) Restriction Fragment Length Polymorphism with gel electrophoresis4

• (C) Gel Electrophoresis5

• Analysis of gel electrophoresis6 and sequence chromatogram (Figure 5, 6, 7)

Resistant (T13) MDA-MB-231 Mitochondria

Hispanic farmworkers of the San Joaquin Valley with polymorphisms in specific

xenobiotic-metabolizing genes have elevated risk of hormone-dependent cancer

4. Restriction

Digest 5. Electrophoresis

6a. RFLP Results 6b. PCR Results

Results

Figure 2. GSTP1-I105V-RFLP

Allele-specific PCR-RFLP of GSTP1-

I105V SNP with BsmAI restriction

endonuclease. A band at 440 bp

represents a wild-type/homozygous

allele. Bands at 440 bp, 228 bp and

212 bp represent a heterozygous

allele, while bands at 228 bp and 212

bp indicate a homozygous

polymorphic allele for that sample.

Figure 1. GSTM1 and GSTT1

Multiplex PCR of GSTM1 and GSTT1.

A band at 215 bp and 480 bp

represent a wild-type/ heterozygous

allele of GSTM1 and GSTT1

respectively. Absence of the bands

indicates null deletion for that gene.

Human Serum Albumen (HSA) is the

positive control.

Figure 4. CYP1A1-T6235C-RFLP

Allele-specific PCR-RFLP of CYP1A1-

T6235C SNP with MspI restriction

endonuclease. A band at 340 bp

represents wild-type/homozygous

allele. Bands at 340 bp, 200 bp and

140 bp represents heterozygous allele,

while bands at 200 bp and 140 bp

indicate a homozygous polymorphic

allele for that sample.

Organochlorines are a common class of pesticides known as endocrine disruptors that can modify the effects of estrogen & testosterone and may act as agonists/antagonists or have mixed effects in the tissue microenvironment.

The pathway for xenobiotic elimination from the body is a multi-step process resulting in excretion of contaminants through urine or bile.

Step 1 involves oxidation by the Phase I P450 cytochrome (CYP) enzyme family, and is typically an activating reaction creating a more polar byproduct.

Step 2 involves conjugation with a ligand by the Phase II glutathione-S-transferase (GST) enzyme family, and is typically a detoxifying reaction.

1. Oragene Kits

3. Sequence

Chromatograph 2. Thermal Cycler

Table 1. SNP Summary.

Twenty-six breast cancer cases and 16 control participants were analysed. Null

genotype for GSTT1 and rs1695, rs1138272, rs1056836 polymorphisms were

associated with increased risk (Odds Ratio > 1) for breast cancer. The

interaction of endosulfan exposure and GSTM1 null (indicative of complete loss

of function to detoxify chemical substrates) gave an O.R. = 13.5, thus a

significant increase in risk for developing breast cancer (data not shown).

However, due to small numbers, neither point estimate was statistically

significant and confidence intervals were very wide.

Figure 3. GSTP1-A114V-RFLP

PCR of GSTP1-A114V SNP select

DNA samples with restriction

endonuclease AciI (5’-C*CGC-3’; 3’-

GGC*G-5’). Bands at 175 bp and 192

bp indicate a wild-type allele. A band

at 367 bp indicates a polymorphic

allele. Presence of all three bands

indicate a heterozygous genotype.

Figure 5. CYP1A1-T6235C Sequence Analysis

Chromatogram showing the coded sequence. Each sample was analyzed for

polymorphism at a single nucleotide position for select xenobiotic-metabolizing

genes. In this example, CYP1A1 (T6235C) gene polymorphism (rs4646903)

was assessed at nucleotide 6235 of the gene locus. Deviation from the CTG

allele indicates the variant allele which has been associated with increased

risk for breast cancer in combination with high serum levels of DDT. The red

arrow indicates the base pair mutation site: CTGCCG.

SNP

designation rs1695 rs1138272 rs1056827 rs1056836

Gene GSTM1 GSTT1

GSTP1

(I105V,

A>G)

GSTP1

(A114V,

C>T)

CYP1B1

(A119S,

G>T)

CYP1B1

(L432V,

C>G)

Genotype

null=nullizygous

WT=wild-type

(positive)

null=nullizygous

WT=wild-type

(positive)

A/A=II

A/G=IV

G/G=VV

C/C=AA

C/T=AV

T/T=VV

G/G=AA

G/T=AS

T/T=SS

C/C=LL

C/G=LV

G/G=VV

O.R. 0.99 2.21 2.79 (A/G)

2.50 (G/G) 2.14 (T/T) 0.77 2.33

95% CI 0.28-3.51 0.39-12.63 0.58-13.3

0.37-16.9 0.38-12.2 0.14-3.70 0.64-8.54