Genotoxicity of Genistein: A Molecular Analysis

Nathan Verlinden, Pramod Mahajan, and Craige WrennDrake University College of Pharmacy and Health Sciences

MethodsGenistein 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.

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.

Introduction

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).

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 p-H2AX 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 Long-Evans rats using protein analysis in rat kidney and liver tissue.

Structure of genistein

ResultsWestern Blot Analysis of H2AX phosphorylation

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

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).

12

11

10

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116 kDa85 kDa

Dimer

Discussion

This research was undertaken to test H2AX phosphorylation and PARP cleavage as markers of potential DNA damage by genistein. Rats were exposed to three different doses of genistein via surgical implantation of genistein-containing pellets in the neck region. Animals receiving similar pellets without genistein served as controls. After 21 days of exposure to genistein, animals were sacrificed, tissue (liver, kidney) harvested , processed and analyzed as described under Methods. Representative results presented in Figures 1 and 2 showed that genistein had no detectable effect on p-H2AX or PARP/PARP cleavage. This indicates that the genistein treatment did not induce apoptosis or double-stranded DNA breaks. The western blot analysis also showed that the antibodies we used showed cross-reactivity to other proteins present in the crude tissue extracts.

Future Studies

The absence of effect on either of the two markers used in the present study may be because there was no DNA damage. However, it is a well-known fact that all living organisms experiencing DNA damage rapidly activate their DNA repair pathways re-instate genomic stability. Consequently, the signaling mechanisms/markers (e.g. H2AX phosphorylation and PARP cleavage) are also reinstated to the state prior to DNA damage. Given the lengthy time frame ( 21 days ), and the transient nature of the signaling mechanisms, it is possible that we missed the window during which we could have detected the modified markers. Clearly, further studies are needed to confirm this contention. Technical issues such as use of high-specificity antibodies, use of nuclear extracts (where the markers proteins are enriched) and/or choice of additional markers for DNA damage (physico-chemical analysis of DNA samples) will be considered. Finally, use of cell cultures as a model system would allow us to precisely manipulate parameters such as exposure time. These studies are currently underway in our laboratory.

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