Genotoxicity of Genistein: A Molecular Analysis
Nathan Verlinden, Pramod Mahajan, and Craige Wrenn
Drake University College of Pharmacy and Health Sciences
Introduction
Methods
Genistein is a phytoestrogen commonly found in soy
products. In recent years, high intakes of soy in Asian diets have
been associated with a decreased incidence of breast cancer
(1). Isoflavones, a sub-class of phytoestrogens that includes
genistein, are thought to confer this chemopreventative effect.
Genistein may do exert its chemopreventative effects by a
number of different mechanisms. First, genistein may be
inhibiting tyrosine kinase acitivity, an important activator of
proliferative pathways (2). Genistein can also act as an
antioxidant, preventing DNA damage (3). Thirdly, genistein can
bind to estrogen receptors to inhibit estrogen receptor
proliferative pathways (4). Lastly, genistein is known to inhibit
the enzyme topoisomerase II leading to double-stranded
breaks in DNA ultimately causing programmed cell death or
apoptosis (5).
Genistein Administration: Adult Long-Evans rats (350-400 g, Charles River, Wilmington, MA) are
anaesthetized by intraperitoneal injection of a mixture of ketamine (3 mg/kg body weight) and
xylazine (2 mg/kg body weight) in saline vehicle (0.1 ml injection volume). The nape of the neck is
shaved with clippers and the skin is disinfected with ethanol. A 3-4 cm incision is made parallel
and lateral to the midline. Using a blunt surgical probe, a small pocket is made between the skin
and muscle medial to the incision for the subcutaneous implantation of the time release pellets.
Rats are implanted with 1-3 pellets (release rate 21 days, Innovative Research of America,
Sarasota, FL) containing 50 mg of genistein. Rats in the 50 mg dose group are implanted with 1
pellet, rats in the 100 mg dose group are implanted with 2 pellets, and rats in the 150 mg dose
group are implanted with 3 pellets. Rats in the control group are implanted with 1-3 placebo
pellets that do not contain genistein. The number of rats per group is 8-10. After implantation of
the pellets, the incision is closed with wound clips and the rat is allowed to recover from
anesthesia under a heat lamp. At the end of the 21-day release period, the rats are sacrificed by
overdose of pentobarbital .Tissue samples are dissected from brain, kidney, and liver and
processed as described below or stored at -80°C for further analysis.
Toposiomerase II inhibition can destroy cancer cells, but
can also harm normal cells. Indeed, therapeutic
topoisomerase II inhibitors are associated with an increase risk
of leukemia (5). However, this genotoxic potential of genistein
on normal cells has not been well established. Since the
consumption of genistein containing products is on the rise in
recent years due to an increase in vegetarian/vegan diets, the
introduction of soy infant formulas, and an increase in soy food
products on the market, it is important to ascertain possible
genotoxic effects of genistein on tissue (6, 7).
This study looks at Poly(ADP-ribose) Polymerase (PARP)
and phophorylated H2AX (p-H2AX) protein levels to determine
genistein genotoxicity. PARP is a nuclear enzyme that signals
when DNA damage is present and helps to recruit repair
complexes to the site of damage (8). PARP inactivation by
cleavage is a tell-tale sign of apoptosis (8). H2AX is a histone
protein that helps to compact DNA (9). Phosphorylation of
H2AX at a serine amino acid 139 is a ubiquitous signal for
double-stranded DNA breaks (9). In this study, PARP and pH2AX will serve as signals of apoptosis and double-stranded
DNA breaks, respectively, caused by genistein. This study aims
to analyze genotoxicity caused by in vivo exposure to LongEvans rats using protein analysis in rat kidney and liver tissue.
Structure of genistein
Protein Extraction Preparation: One gram of frozen tissue was ground up into a powder using a
motor and pestle that was stored at -80°C over night. The powdered tissue was quickly transferred
to a centrifuge vial. Two mL of Tissue- Protein Extraction Reagent (T-PER) per gram of tissue powder
was added and the mixture was homogenized on ice for 5 minutes while alternating 30 seconds
on and 30 seconds off cycles. The homogenized mixture was then quickly vortexed and
centrifuged at 10,000x g for five minutes at 4°C. The supernatant was pipetted off and stored at 80oC. The cell debris pellet was also stored separately.
Bradford Protein Analysis: A series of dilutions of the protein samples were prepared at ratios of
1:10, 1:50, and 1:100. The dilutions were analyzed using the Bradford micro-assay which uses the
dye-binding method to measure absorbance of protein containing solution at 595nm. The
absorption values recorded were used to calculate the protein concentration of the solutions.
Bovine serum albumin (BSA) was used as a standard. A standard curve for protein
concentration and absorption at 595 nm ( A595) was constructed using BSA samples of known
protein concentrations between 1-10 μg/ml. The value for the slope of the straight line was used to
determine the amount of protein in each sample. These values were used to analyze equal
amounts of protein from each tissue sample using Sodium dodecyl sulfate - polyacrylamide gel
electrophoresis (SDS-PAGE) followed by the western blotting technique.
Polyacrylamide Gel Electrophoresis in presence of SDS and β-mercaptoethanol: Appropriate
volumes of each protein extraction were diluted so equal amounts of protein will be loaded into
each well of the gel. Protein samples were combined with an equal volume of 2x SDS-sample
buffer containing 2% SDS and 1 M β-mercaptoethanol as the reducing agent. The mixtures were
vortexed and quickly spun before they were heated at 95°C for five-six minutes. The samples were
quickly spun again after heating and were loaded onto a 4-20% Tris-glycine gel. Along with the
protein samples from the tissue other controls were also load onto the gel to provide a negative
and positive control for comparison. The gel apparatus was filled with 1% Tris-glycine running
containing 0.1 % SDS and electrophoresis was carried out at 150 volts for 90 minutes. The gel was
removed from the plastic cassette and protein was transferred on to a PVDF membrane using the
Western transfer protocol described below.
Western Blot: Immediately following the electrophoresis, the gel was washed with Tris-glycine
transfer buffer containing 20% methanol. Sponge pads were soaked in cold ( 4oC) 1x transfer
buffer containing SDS until the assembly of the transfer box. Two filter papers were briefly rinsed in
water and 1x transfer buffer. The PVDF membrane which is what the proteins will be transferring to
from the gel is highly hydrophobic so before soaking in water and transfer buffer it needed to be
rinsed in methanol. First three sponge pads were place in the box followed by a filter paper. Next
the gel was carefully placed on the filter paper. The PVDF membrane was place on the gel
carefully making sure that there were no air bubbles between any of the layers to disrupt the flow
of current and ultimately the transfer of proteins. The second filter paper was placed on the
membrane followed by three more sponge pads. The box was closed and place in the running
apparatus where the inner chamber was filled with the 1x transfer buffer along with 1 mL of 10%
SDS. The outer chamber was filled with 1x transfer buffer and the protein transfer was performed at
25 volts for 2 hours.
Protein Detection: The blot is soaked in a non-specific blocking agent, Blotto, in tris-buffered saline
with tween (TBST), to saturate the areas on the blot where there is no protein so only specific
binding happens through the detection process. After the blot is soaked for at least 20 minutes the
blot is washed for 10 minutes in TBST. Rabbit polyclonal IgG primary antibodies against PARP and
p-H2AX (Santa Cruz Biotechnologies) were used at a dilution of 1:1000 pre-prepared in TBST
containing 4% Blotto. The membrane was exposed to the primary antibody solution for an hour.
After this step, the blot is washed 3 times each time for 10 minutes in TBST. The blot is then exposed
to a donkey polyclonal secondary antibody (Abcam Biotechnologies) which is an antibody that is
specific the primary antibody. The secondary antibody is linked with horse-radish peroxidase, an
enzyme that catalyzes a chemi-luminescent reaction. After one hour incubation with the
secondary antibody, the blot is washed 3 times each time for 10 minutes in TBST. After the washing
after the secondary antibody a luminescence solution was carefully dropped onto the blot. The
solution was prepared by adding 50μL of solution B to 2mL of solution A that are provided in the
GE ECL kit. The blot was then placed on a glass screen so the imager could expose the blot. The
standard exposure times for the blots were typically between 30 seconds to 5 minutes. The
glowing bands were captured and were able to be analyzed from the image.
Results
Discussion
Western Blot Analysis of H2AX phosphorylation
1 2
3
4
5
6
7
8
9 10 11 12
Figure 1 : Analysis of expression of p-H2AX in rat kidney and liver. Electrophoresis and Western blotting was
performed as described under Methods. Lane 1: SB+ marker, Lane 2: Jurkat (etoposide induced) extract, Lane 3:
Jurkat non-induced extract, Lane 4: Liver tissue 4.7, Lane 5: Liver tissue 3.8, Lane 6: Liver tissue 2.5, Lane 7: Liver
tissue 1.4, Lane 8: Kidney tissue 4.4, Lane 9: Kidney tissue 3.2, Lane 10: Kidney tissue 2.3, Lane 11: Kidney tissue 1.8,
Lane 12: SB marker. The orange circle denotes a positive detection of p-H2AX.
As expected, the etoposide-treated Jurkat cell extracts exhibit a substantially higher level of p-H2AX
protein than the non-treated Jurkat cells. There were no significant signs of p-H2AX in either the kidney
or liver tissue. In an attempt to detect even low levels of p-H2AX in the rat kidney and liver samples, the
blot was overexposed purposely . This resulted in the very high non-specific background, but failed to
show any p-H2AX protein in the kidney and liver samples.
Western Blot Analysis of PARP cleavage
Dimer
116 kDa
85 kDa
1
2
3
4
5
This research was undertaken to test H2AX ph
and PARP cleavage as markers of potential DNA
genistein. Rats were exposed to three different d
genistein via surgical implantation of genistein-con
in the neck region. Animals receiving similar pelle
genistein served as controls. After 21 days of exp
genistein, animals were sacrificed, tissue (liver, kidn
processed and analyzed as described under Meth
Representative results presented in Figures 1 and 2
genistein had no detectable effect on p-H2AX or
cleavage. This indicates that the genistein treatm
induce apoptosis or double-stranded DNA breaks
blot analysis also showed that the antibodies we u
cross-reactivity to other proteins present in the cru
extracts.
Future Studies
The absence of effect on either of the two m
the present study may be because there was no D
However, it is a well-known fact that all living orga
experiencing DNA damage rapidly activate their
pathways re-instate genomic stability. Consequen
signaling mechanisms/markers (e.g. H2AX phosph
PARP cleavage) are also reinstated to the state pr
damage. Given the lengthy time frame ( 21 days
transient nature of the signaling mechanisms, it is
missed the window during which we could have d
modified markers. Clearly, further studies are need
this contention. Technical issues such as use of hig
antibodies, use of nuclear extracts (where the ma
are enriched) and/or choice of additional marke
damage (physico-chemical analysis of DNA samp
considered. Finally, use of cell cultures as a mode
allow us to precisely manipulate parameters such
time. These studies are currently underway in our
References
1.
2.
Figure 2. Analysis of PARP cleavage in rat kidney samples. Electrophoresis and Western blotting
was performed as described under Methods. Lane 1: HeLa nuclear extract, Lane 2: Kidney
tissue 1.3, Lane 3: Kidney tissue 2.3, Lane 4: Kidney tissue 4.4, Lane 5: SB markers. Circle denotes
a positive detection of PARP. The green arrow indicates the native, 116 kDa PARP band and
the red arrow indicates the 85 kDa cleavage product of PARP. The dimerized form of PARP,
indicated by the black arrow is also detected in HeLa extracts .
The HeLa cells expressed high levels of PARP and also showed evidence of PARP cleavage in
response to apoptosis occurring in cultured cells. None of the rat kidney extracts showed
appreciable amounts of PARP, either native or cleaved, despite overexposure of the blot.
Similar results were obtained when liver samples were analyzed ( data not shown).
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