Download The Effects of Glutatione-S-Transferase (GST

The Effects of Glutathione-S-Transferase Polymorphisms on Sulforaphane Metabolism
Jeannie Allen1, Anna Hsu1, Lauren Atwell1, 2, John D. Clarke1, Deborah Bella1, Emily Ho1, 2
1
School of Biological and Population Health Sciences, 2Linus Pauling Institute, Oregon State University,
Corvallis, OR 97331
Abstract: Isothiocyanates are chemo-preventative compounds that are converted from their precursors, the
glucosinolates, which are found in cruciferous vegetables. Sulforaphane (SFN) is an isothiocyanate that has
recently gained interest as having the potential to decrease individual cancer risk. After conversion from its
glucosinolate precursor, glucoraphanin, SFN is further metabolized via the mercapturic acid pathway and
there is debate as to whether SFN is the biologically active compound or if SFN metabolites are responsible
for the anti-cancer effects. The first enzyme to act on SFN is a glutathione-S-transferase (GST), which
exhibits polymorphisms in a significant percentage of the population. In this study, subjects consumed
broccoli sprouts that contain high levels of glucoraphanin, the SFN precursor. SFN and metabolite levels
were measured by mass spectrometry in the urine and plasma and metabolite levels were compared between
wild type and polymorphic individuals for GSTA1, GSTM1, GSTP1 (codons 105 and 114) and GSTT1. No
significant differences were found in GSTA1, GSTP1 or GSTT1 but GSTM1 null individuals exhibited lower
levels of SFN-GSH and SFN-CG in urine.
Introduction: Glutathione-S-transferases (GSTs) are important enzymes that catalyze reactions to detoxify
carcinogens from the body and hence play a critical role in cancer prevention. GSTs are also involved in the
metabolism of sulforaphane (SFN), a chemo-preventative phytochemical in cruciferous vegetables. GSTs
perform the first step in SFN metabolism by conjugating a glutathione (GSH) group to SFN. The metabolism
of SFN is a key factor in its chemo-preventative benefits as both SFN (Clarke et al, 2008; Higdon et al, 2007;
Ho et al, 2009) and subsequent metabolites have been shown to act chemo-preventatively (Ho et al 2009,
Myzak et al, 2004). Polymorphisms in the GST genes are highly prevalent; screening for GST
polymorphisms in a healthy population showed that 35 out of 36 subjects had at least one GST
polymorphism. Polymorphisms are commonly associated with a decrease in activity but this decrease in
activity demonstrates substrate-dependent variability (Di Pietro et al, 2010; Ginsberg et al, 2009; Navarro et
al, 2009; Steck et al, 2006; Ping et al, 2006).
Diets high in cruciferous vegetables such as broccoli and cauliflower are associated with a lower risk
of many common cancers (Clarke et al, 2008). Specifically, one family of compounds called isothiocyanates
have been proposed to be the active phytochemicals derived from cruciferous vegetables with anti-cancer
properties. Isothiocyanates such as SFN are metabolized from their precursor compounds, the glucosinolates
[Fig. 1], which are present in plants. The conversion of glucosinolates to isothiocyanates occurs in the body
after consumption by the enzyme myrosinase; exposure of glucosinolates to myrosinase occurs when the
plant is chewed or chopped. Myrosinase is also produced by gastrointestinal flora in much smaller amounts.
SFN is further metabolized in the mercapturic acid pathway to produce potentially biologically active
compounds [SFN-Glutathione (SFN-GSH), SFN- cysteinylglycine (SFN-CG), SFN-cysteine (SFN-Cys) and
SFN-N-acetylcysteine (SFN-NAC)] [Fig.2].
There are 7 isozymes of GST in its super-family and polymorphisms are present in at least one GST
in 90% of the population (Ginsberg et al, 2009). Currently the effects of many GST polymorphisms on SFN
bioavailability and metabolite production in humans have not been extensively studied. Thus, the goal of this
project is to investigate whether polymorphisms in GSTA1, GSTM1, GSTP1 (codons 105 and 114) and
GSTT1 affect levels of SFN and SFN metabolites in the blood and urine. This will help to ascertain if
individuals with GST polymorphisms have different SFN responses due to their genotypes.
Methods
Intervention: After abstaining from cruciferous vegetables for 7 days, study subjects consumed 40 grams of
broccoli sprouts containing approximately 150 µ(micro)mol of glucoraphanin.
Sample Collection: Urine and blood samples were collected from the study described above. When urine
was collected, the volume was measured and recorded before 30ml was extracted and mixed with 3ml
trifluoroacetic acid (TFA) to stabilize SFN metabolites. It was then transferred using urine transfer caps and
saved in separate test tubes at -80° C.
Fasting blood samples were collected before consumption of broccoli sprouts. Blood samples were also
collected after 3, 6, 12, 24 and 48 hours. 1.5 ml of whole blood was transferred and centrifuged at room
temperature for a minute at full speed to separate red blood cells and plasma. 700 μ(micro)l of plasma was
extracted and added to 70 µ(micro)l TFA. The sample was then vortexed and stored at -80° C.
GST Polymorphism Analysis: GSTA1 is located on chromosome 6p12 and was analyzed by polymerase
chain reaction restriction fragment length polymorphism (PCR-RFLP) as described by Ping et al, 2006.
GSTP1 at codon 105 was determined by PCR-RFLP as described by Oliviera et al, 2010. GSTP1 at codon
114 was examined by PCR-RFLP as described by Lee et al, 2004. The GSTM1 polymorphism at codon
1p13.3 and the GSTT1 polymorphism at codon 22q11.2 were analyzed together by multiplex PCR as
described by Oliviera et al, 2010.
Measurement and Analysis of Metabolites: Metabolite levels in urine and plasma were measured by mass
spectrometry. Urine was diluted 1:10 with 0.1% (v/v) formic acid in water. Plasma processing involved
addition of cold TFA (0° C) at 10% (v/v) to plasma that had been pre-chilled on ice. After five minutes on
ice, the cloudy suspension was centrifuged at 16,000 x g for 5 min to pellet the proteins and recover
metabolites in the supernatant. Supernatant was injected directly (10 μ(micro)l) for UHPLC-MS/MS
analysis. They were then analyzed using two way ANOVA.
Results: Table 1 shows the total SFN and metabolite levels in the urine. These levels include the total sum
of SFN and all of its metabolites [SFN-Glutathione (SFN-GSH), SFN- cysteinylglycine (SFN-CG), SFNcysteine (SFN-Cys) and SFN-N-acetylcysteine (SFN-NAC)]. There were no significant differences in total
SFN excretion between genotypes. There was only one individual who was polymorphic at codon 114 of
GSTP1 so this genotype could not be statistically analyzed. Table 2 shows the total peak SFN metabolite
levels (sum of SFN and all metabolites) found in the plasma. Similar to urine, there were no significant
differences between genotypes in total peak SFN levels. Thus GST polymorphisms did not appear to have
any impact on total uptake and excretion of SFN.
The effects of GST polymorphisms on individual SFN metabolites were also analyzed separately. No
genotype differences were seen in GSTP1 at codon 105 or GSTA1 and GSTT1 in SFN, SFN-GSH, SFN-CGSFN-Cys or SFN-NAC in plasma or urine. However, for GSTM1 null, there was an overall trend of lower
SFN-GSH and SFN-CG [Fig. 3 and 4]. Thus, GST polymorphisms had no effect on total SFN levels in
plasma or urine, but upon analysis of individual metabolites GSTM1 null genotypes had lower SFN-GSH and
SFN-CG.
Discussion: SFN and its subsequent metabolites are important dietary chemo-preventative agents. Due to
their accessibility and low cost, cruciferous vegetables have the potential to decrease cancer prevalence as
simple additions to a healthy diet. The goal of this project was to examine the effect of polymorphisms in
GSTA1, GSTP1 (codons 105 and 114), GSTM1 and GSTT1 on conversion of SFN to its metabolites.
The four polymorphisms that could be statistically analyzed (GSTA1, GSTP105, GSTM1 and GSTT1)
exerted no significant effect on overall SFN conversion to its metabolites. However, GSTM1 null individuals
showed decreased urine concentrations of SFN-GSH and SFN-CG, the two direct products of GST action.
Our results suggest that inter-individual variability in SFN metabolism may be due, at least in part, to
polymorphisms in GSTs, especially GSTM1. Individuals who are GSTM1 null showed decreased levels of
SFN-GSH and SFN-CG in the urine indicating that they may metabolize SFN less efficiently. Therefore, it is
important to learn more about this polymorphism and to what extent it may decrease SFN metabolism.
Figure 1. Conversion of the glucosinolate, glucoraphanin, to sulforaphane (SFN), an isothiocyanate, by the
enzyme myrosinase.
Figure 2. Conversion of sulforaphane (SFN) to sulforaphane-glutathione (SFN-GSH) by a glutathione-stransferase (GST), sulforaphane cysteinylglycine (SFN-CG) by γ-glutamyltranspeptidase (GTP),
sulforaphane cysteine (SFN-Cys) by cysteinylglycinase (CGase) and sulforapane-N-acetylcysteine (SFNNAC) by N-acetyltransferase (NAT) in the mercapturic acid pathway.
Table 1. Total SFN and metabolite levels in urine after broccoli sprout consumption.
Table 2. Total SFN and metabolite levels in plasma after broccoli sprout consumption.
Figure 3. GSTM1 null shows decreased levels of SFN-GSH in urine after broccoli sprout consumption. Pvalues: genotype-0.0262, time-0.0001, interaction-0.1998.
Figure 4. GSTM1 null shows decreased levels of SFN-CG in the urine after broccoli sprout consumption. Pvalues: genotype-0.0081, time-0.0003, interaction-0.1043
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