The Effects of Glutathione-S-Transferase Polymorphisms on Sulforaphane Metabolism

By Jeannie AllenMentored by Dr. Emily Ho

Biological and Population Health Sciences

Cancer Background Cancer is the second leading cause of death in

the US [Centers for Disease Control and Prevention]. One in two men and one in three women will get some form of cancer in their lifetimes [American Cancer Society].

The total cost of cancer in 2010 was over 124 billion dollars and is projected to be 158 billion in 2020 [National Cancer Institute].

Do Not Fear, Broccoli Is Here!

Epidemiological studies have shown that an increase in cruciferous vegetable intake is correlated with reduced cancer risk [Clarke et al].

Isothiocyanates are one of the major anti- cancer bioactive compounds found in cruciferous vegetables, such as broccoli.

Sulforaphane is an isothiocyanate found in cruciferous vegetables such as broccoli, broccoli sprouts, cauliflower and Brussels sprouts.

Sulforaphane has been shown to be an effective chemoprotective agent in vitro and in vivo by:› selectively inducing apoptosis in cancer cells› slowing tumor growth › Inhibiting HDAC activity [Ho et al]› Regulating phase I and II enzymes [Clarke et al]

Metabolism of sulforaphane in humans is not well known.

Sulforaphane

Absorption and Bioavailability of Sulforaphane

Isothiocyanates are metabolized from their precursors, the glucosinolates. [Figure: Clarke, et.al.]

Myrosinase is found in cruciferous plants in the cell wall or in the human gastrointestinal microflora.

(Glucoraphanin) (Sulforaphane)

SFN is metabolized via the Mercapturic Acid pathway

The glutathione-s-transferase (GST) genes encode key enzymes (the GSTs) that are involved in the metabolism of sulforaphane.

SFN = Sulforaphane SFN-GSH = Sulforaphane Glutathione SFN-CG = Sulforaphane Cysteinylglycine SFN-Cys = Sulforaphane Cysteine SFN-NAC = Sulforaphane-N-Acetylcysteine

Polymorphisms are prevalent among humans with 90% or more of the population being polymorphic in at least one site [Ginsberg et al].

Glutathione-S-Transferases Glutathione-S-transferases are a superfamily of enzymes

that include 7 different isoforms. The forms of the GST gene that were studied are GSTA1

(α), GSTP1 (π), GSTM1 (μ) and GSTT1 (θ) [ Prevalence distribution from Di Pietro et al, Ginsberg et al and Steck et al].

Caucasians

Asians African Americans

Mexican Americans

GSTM1 positive 46% 42-55% 79% 59%GSTT1 positive 80% 42-55% 78% 89%GSTP1 105 Ile/Ile 30-55% 44-68% 6-53%GSTP1 105 Ile/Val 34-65% 25-50% 39-80%GSTP1 105 Val/Val 3-14% 2-8% 8-23%GSTP1 114 Ala/Ala 82% 95%GSTP1 114 Ala/Val 18% 5%GSTP1 114 Val/Val 0% 0%GSTA1 68% of the general population is polymorphic

Hypothesis

To find out whether differences in an individual’s genotype in GSTM1, GSTT1, GSTP1 and GSTA1 affect their metabolism and excretion of sulforaphane.

Individuals with polymorphisms of some of the GST genes may have altered metabolism and absorption of sulforaphane.

Objective

Analysis of GSTP1 Polymorphism

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)

GSTP1 is polymorphic at two sites: codon 105 and 114 Different polymorphisms are associated with higher or

lower activity in a substrate dependent manner.

Codon 105 Codon 114

GSTP*A (wild type)

Isoleucine (Ile) Alanine (Ala)

GSTP*B Valine (Val) AlanineGSTP*C Valine ValineGSTP*D Isoleucine Valine

3’ 5’

PCR-RFLP:PCR Amplification of GSTP (105 and 114) and

restriction enzyme digestion

GSTP gene

templatePrimer-forward

Primer-reverse3’

5’

BsmA1 or Aci1 digestion of PCR product

NNNNNNNNNNNNNGTCTCNNNNNNNNNNNNNNNNN

BsmA1

DNA polymerase

Gel Electrophoresis

Gel electrophoresis results of GSTP (105)

200 base pairs100 base pairs

A single band at 176 base pairs indicates homozygosity (GSTP 105 Ile/Ile).

A band at 176 bp, two at 91 and 86 bp indicates that the individual is heterozygous (GSTP 105 Ile/Val).

Two bands at 91 and 86 bp without one at 176 bp indicates variant homozygosity (GSTP 105 Val/Val)

GSTA1 was analyzed in a similar fashion.

Analysis of GSTM1 and GSTT1 polymorphism

GSTM1 and GSTT1 were analyzed using multiplex PCR.

For either gene, a person either expresses the gene (positive) or doesn’t (null).

GSTT 480 bpAlbumin 350 bpGSTM 215 bp

Goal: To examine SFN Metabolites after Broccoli Consumption

Genomic DNA was collected from whole blood for polymorphism analysis at baseline and SFN metabolite levels were determined by mass spectrometry.

Participants gave blood and urine samples after consuming broccoli and alfalfa sprouts

Broccoli sprouts (n=12) alfalfa sprouts (n=4)

Results and Discussion

Positive Null Homozygous wt

Heterozygous

Homozygous variant

GSTA1 - - 4 (25%) 7 (43.75%) 5 (31.25%)GSTP1(105)

- - 11 (68.75%)Ile/Ile

5 (31.25%)Ile/Val

0Val/Val

GSTP1(114)

- - 15 (93.75%)Ala/Ala

1 (6.25%)Ala/Val

0Val/Val

GSTM1 6 (37.5%)

10(62.5%)

- - -

GSTT1 11 (68.75%

)

5(31.25%

)

- - -

Distribution of GST Polymorphisms Among Study Subjects

No significant difference found between genotypes on total metabolites excreted

in urine after broccoli sprouts consumption

Genotype Total amount of SFN metabolites excreted, µmols

Homozygous Wild Type Heterozygous Homozygous

VariantGSTA1 168.5 ± 90.2 151.4 ± 21.1 149.6 ± 35.6GSTP1 (105)

171.6 ± 61.9Ile/Ile

135.1 ± 33.1Ile/Val

N/AVal/Val

GSTP1 (114)

162.7 ± 50.9Ala/Ala

86.3 #

Ala/ValN/A

Val/ValGSTM1 179 ± 69.8 140.2 ± 35GSTT1 165.3 ± 56.4 138.5 ± 36.1

# indicates n=1

No significant differences found between genotypes on total metabolites in plasma

after broccoli sprouts consumption

Genotype Peak concentration of SFN metabolites in plasma, µmols/L

Homozygous Wild Type Heterozygous Homozygous

VariantGSTA1 2.4 ± 0.9 2.4 ± 0.2 2.3 ± 0.8GSTP1 (105)

2.1 ± 0.5Ile/Ile

2.7 ± 0.8Ile/Val

n/aVal/Val

GSTP1(114)

2.4 ± 0.7Ala/Ala

1.4 #

Ala/Valn/a

Val/ValGSTM1 2.0 ± 0.6 2.5 ± 0.8GSTT1 2.4 ± 0.6 2.3 ± 0.9# indicates n=1

GSTM1 positive showed increased excretion of SFN-GSH after broccoli

sprouts consumption

p-value

Genotype 0.0262

Time (hour)

0.0001

Interaction 0.1998

GSTM1 positive showed increased excretion of SFN-CG after broccoli

sprouts consumption

p-value

Genotype 0.0081

Time (hour)

0.0003

Interaction 0.1043

Conclusion The GSTA1, GSTP1, GSTM1 and GSTT1

polymorphisms that were examined have no effects on overall sulforaphane metabolism and excretion.

Individuals who are GSTM1 null excrete less SFN-GSH and SFN-CG, suggesting that they may metabolize sulforaphane less efficiently.

The National Cancer Institute recommends 5-9 servings of fruits and vegetables daily.

Cohort studies suggest a weekly consumption of at least 5 servings of cruciferous vegetables to gain optimal chemopreventative benefits of sulforaphane [Higdon].

Acknowledgements Dr. Emily Ho, Ph.D. Dr. Anna Hsu, Ph.D. Dr. John Clarke, Ph.D. Ho lab

› Karin Hardin› Carmen Wong, Ph.D.› Laura Beaver, Ph.D. › Lauren Atwell, M.S., R.D.

Mass Spectrometry Facility› Dr. Fred Stevens, Ph.D.› Jeff Morré

Dr. Kevin Ahern, Ph.D. Funding sources: Howard Hughes Medical Institute Summer

Fellowship (HHMI) and OSU’s Environmental Health Sciences Center (EHSC).

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