
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].
 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.

Isothiocyanates are metabolized from their
precursors, the glucosinolates. [Figure: Clarke, et.al.]
(

Glucoraphanin)
(
Sulforaphane)
Myrosinase is found in cruciferous plants in the cell
wall or in the human gastrointestinal microflora.







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 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
GSTM1 positive
GSTT1 positive
GSTP1 105 Ile/Ile
46%
80%
30-55%
42-55%
42-55%
44-68%
African
Americans
79%
78%
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
Mexican
Americans
59%
89%
68% of the general population is polymorphic
Individuals with polymorphisms of some of the GST
genes may have altered metabolism and
absorption of sulforaphane.
To find out whether differences in an individual’s
genotype in GSTM1, GSTT1, GSTP1 and GSTA1 affect
their metabolism and excretion of sulforaphane.
Codon 105
Codon 114
GSTP*A
(wild type)
Isoleucine (Ile)
Alanine (Ala)
GSTP*B
Valine (Val)
Alanine
GSTP*C
Valine
Valine
GSTP*D
Isoleucine
Valine
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.

Primer-forward
DNA polymerase
3’
5’ template
5’
GSTP gene
BsmA1
Primer-reverse
BsmA1 or Aci1 digestion of PCR
product
NNNNNNNNNNNNNGTCTCNNNNNNNNNNNNNNNNN
200 base pairs
100 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.


GSTM1 and GSTT1
were analyzed using
multiplex PCR.

For either gene, a
person either
expresses the gene
(positive) or doesn’t
(null).
GSTT 480 bp
Albumin 350 bp
GSTM 215 bp
Participants gave blood and
urine samples after
consuming broccoli and
alfalfa sprouts
Broccoli sprouts (n=12)
alfalfa sprouts (n=4)
Genomic DNA was collected from whole blood for
polymorphism analysis at baseline and SFN metabolite
levels were determined by mass spectrometry.
Distribution of GST Polymorphisms Among Study Subjects
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
0
Val/Val
GSTP1(114)
-
-
15 (93.75%)
Ala/Ala
1 (6.25%)
Ala/Val
0
Val/Val
GSTM1
6
(37.5%)
10
(62.5%)
-
-
-
GSTT1
11
(68.75%)
5
(31.25%)
-
-
-
Genotype
Total amount of SFN metabolites excreted,
µmols
Homozygous
Wild Type
Heterozygous
Homozygous
Variant
GSTA1
168.5 ± 90.2
151.4 ± 21.1
149.6 ± 35.6
GSTP1 (105)
171.6 ± 61.9
Ile/Ile
135.1 ± 33.1
Ile/Val
N/A
Val/Val
GSTP1 (114)
162.7 ± 50.9
Ala/Ala
86.3 #
Ala/Val
N/A
Val/Val
GSTM1
179 ± 69.8
140.2 ± 35
GSTT1
165.3 ± 56.4
138.5 ± 36.1
# indicates n=1
Genotype
Peak concentration of SFN metabolites in
plasma, µmols/L
Homozygous
Wild Type
Heterozygous
Homozygous
Variant
GSTA1
2.4 ± 0.9
2.4 ± 0.2
2.3 ± 0.8
GSTP1
(105)
2.1 ± 0.5
Ile/Ile
2.7 ± 0.8
Ile/Val
n/a
Val/Val
GSTP1
(114)
2.4 ± 0.7
Ala/Ala
1.4 #
Ala/Val
n/a
Val/Val
GSTM1
2.0 ± 0.6
2.5 ± 0.8
GSTT1
2.4 ± 0.6
2.3 ± 0.9
# indicates n=1
p-value
Genotype 0.0262
Time (hour) 0.0001
Interaction 0.1998
p-value
Genotype 0.0081
Time (hour) 0.0003
Interaction 0.1043

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].
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).

1.
2.
3.
4.
5.
6.
7.
8.
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