EP3148560A2 - USE OF SECOISOLARICIRESINOL DIGLUCOSIDES (SDGs) AND RELATED COMPOUNDS FOR PROTECTION AGAINST RADIATION AND CHEMICAL DAMAGE - Google Patents
USE OF SECOISOLARICIRESINOL DIGLUCOSIDES (SDGs) AND RELATED COMPOUNDS FOR PROTECTION AGAINST RADIATION AND CHEMICAL DAMAGEInfo
- Publication number
- EP3148560A2 EP3148560A2 EP15799095.3A EP15799095A EP3148560A2 EP 3148560 A2 EP3148560 A2 EP 3148560A2 EP 15799095 A EP15799095 A EP 15799095A EP 3148560 A2 EP3148560 A2 EP 3148560A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- sdg
- subject
- radiation
- exposed
- bioactive ingredient
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Classifications
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/075—Ethers or acetals
- A61K31/085—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon
- A61K31/09—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon having two or more such linkages
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
Definitions
- compositions and methods for radioprotection and radiation mitigation and for chemoprevention such as from carcinogen-induced lung cancer and mesothelioma or from hypochlorite ions, using secoisolariciresinol diglucoside (SDG), secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL), stereoisomers thereof, metabolites thereof, and analogs thereof.
- SDG secoisolariciresinol diglucoside
- SECO secoisolariciresinol
- ED enterodiol
- EL enterolactone
- Ionizing radiation produces a wide range of deleterious effects in living organisms. Humans are exposed to radiation as an occupational hazard, during diagnostic and therapeutic radiographic procedures, when using electronic devices, from background radiation of nuclear accidents, during air and space travel, as well as from prolonged exposure to the sun (e.g. , sun bathers or outdoor workers). Exposure to natural radiation can occur in many forms: natural resources such as air, water, and soil may become contaminated when they come in contact with naturally-occurring, radiation-emitting substances (radionuclides); radon is one such common source of natural radiation. Current global developments have additionally established terrorism as a dangerous means by which potentially large numbers of people can be exposed to lethal amounts of radiation. It is, therefore, of high importance to identify agents that can be administered before and during exposure to radiation (i.e., radioprotective agents), and as treatment after radioactive exposure (i.e., radiation mitigators).
- radioprotective agents agents that can be administered before and during exposure to radiation
- radioactive exposure i.e., radiation mit
- lung cancer is the leading cause of cancer mortality in the United States.
- novel targeted therapeutic agents improved staging and surgical techniques, and increased utilization of concomitant chemoradiation therapy for locally advanced lung cancer there has only been a minimal decrease of overall mortality rates (Tan & Spivack (2009) Lung Cancer 65:129-137).
- Cancer chemoprevention has been defined as the use of dietary and pharmacological intervention with specific natural or synthetic agents designed to prevent, suppress, or reverse the process of carcinogenesis before the development of malignancy (Hong & Sporn, (1997). Science 278: 1073-1077).
- MM malignant mesothelioma
- lung cancer Carbone & Yang (2012). Clin Cancer Res 18:598-604; Neri et al. (2012). Anticancer Res 32:1005-1013
- MM is a highly aggressive cancer that arises from the mesothelial cells of the pleura and peritoneum with a median survival of about 1 year (Sterman et al. (2005).
- Chemoprevention of cancer aims to prevent, arrest, or reverse either the initiation phase of carcinogenesis or the progression of neoplastic cells to cancer.
- this definition sounds simple, it has been very difficult to find effective chemopreventive agents.
- the mechanisms by which carcinogens induce cancer usually involve multiple mechanisms, making efficacy challenging and requiring an agent with multiple activities.
- agents i.e., chemopreventive agents
- carcinogens or other harmful chemical agents such as chemical warfare agents, chlorine and hypochlorite ions and other harmful toxicants.
- the invention relates to a method for protecting a biomolecule (such as genetic material like a nucleic acid, a protein or a lipid), a cell, or a tissue, from radiation damage in a subject in need thereof, the method comprising: administering to said subject an effective amount of flaxseed, its bioactive ingredient, degradant or a metabolite thereof.
- Administration to said subjects encompasses administration prior to, during and after exposure to damaging radiation exposure.
- the time prior, during and post could be seconds, minutes, hours, days, weeks, months or even years.
- the bioactive ingredient encompasses secoisolaricirecinol diglucoside (SDG), secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or a combination thereof.
- SDG secoisolaricirecinol diglucoside
- SECO secoisolariciresinol
- ED enterodiol
- EL enterolactone
- the invention in another aspect, relates to a method for treating or preventing radiation damage in a subject who has been or will be exposed to radiation, the method comprising: administering to said subject an effective amount of at least one bioactive ingredient, wherein said bioactive ingredient comprises secoisolaricirecinol diglucoside (SDG), secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or a combination thereof.
- SDG secoisolaricirecinol diglucoside
- SECO secoisolariciresinol
- ED enterodiol
- EL enterolactone
- the invention relates to a method for protecting biomolecule (such as genetic material like a nucleic acid, a protein or a lipid), a cell, or a tissue, from radiation damage resulting from accidental radiation exposure in a subject in need thereof, the method comprising: administering to said subject an effective amount of flaxseed, its bioactive ingredient, degradant or a metabolite thereof.
- biomolecule such as genetic material like a nucleic acid, a protein or a lipid
- cell or a tissue
- the invention relates to a method for protecting biomolecule (such as genetic material like a nucleic acid, a protein or a lipid), a cell, or a tissue, from radiation damage resulting in aging.
- biomolecule such as genetic material like a nucleic acid, a protein or a lipid
- the invention relates to a method for protecting a biomolecule (such as genetic material like a nucleic acid, a protein or a lipid), a cell, or a tissue from damage resulting from exposure to chemical carcinogens and toxicants both natural and synthetic.
- the invention relates to a method for protecting a biomolecule (such as genetic material like a nucleic acid, a protein or a lipid), a cell, or a tissue, from radiation damage resulting from radiation therapy for cancer treatment in a subject in need thereof, the method comprising: administering to said subject an effective amount of flaxseed, its bioactive ingredient, degradant or a metabolite thereof.
- a biomolecule such as genetic material like a nucleic acid, a protein or a lipid
- a cell such as a protein or a lipid
- a tissue comprising: administering to said subject an effective amount of flaxseed, its bioactive ingredient, degradant or a metabolite thereof.
- the invention relates to a method for protecting a biomolecule (such as genetic material like a nucleic acid, a protein or a lipid), a cell, or a tissue from radiation damage in a subject in need thereof, the method comprising: administering to said subject an effective amount of at least one bioactive ingredient, wherein said bioactive ingredient comprises secoisolariciresinol diglucoside (SDG), secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or a combination thereof.
- the radiation damage results from accidental radiation exposure.
- the radiation damage results from radiation therapy for cancer (e.g. , lung cancer) treatment.
- the invention relates to a method for preventing radiation induced damage to a biomolecule (such as genetic material like a nucleic acid, a protein or a lipid), a cell, or a tissue, in a subject in need thereof, the method comprising: administering to said subject an effective amount of flaxseed, its bioactive ingredient, or a metabolite thereof.
- a biomolecule such as genetic material like a nucleic acid, a protein or a lipid
- the invention relates to a method for preventing radiation induced damage to a biomolecule (such as genetic material like a nucleic acid, a protein or a lipid), a cell, or a tissue, in a subject in need thereof, the method comprising: administering to said subject an effective amount of at least one bioactive ingredient, wherein said bioactive ingredient comprises secoisolariciresinol diglucoside (SDG), secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or a combination thereof.
- SDG secoisolariciresinol diglucoside
- SECO secoisolariciresinol
- ED enterodiol
- EL enterolactone
- the invention relates to a method for protecting a biomolecule (such as genetic material like a nucleic acid, a protein or a lipid), a cell, or a tissue, from radiation damage in a cell, the method comprising contacting said cell with an effective amount of at least one bioactive ingredient, wherein said bioactive ingredient comprises secoisolariciresinol diglucoside (SDG), secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or a combination thereof.
- SDG secoisolariciresinol diglucoside
- SECO secoisolariciresinol
- ED enterodiol
- EL enterolactone
- the invention in another aspect, relates to a method for protecting a biomolecule (such as genetic material like a nucleic acid, a protein or a lipid), a cell, or a tissue, from carcinogen damage in a subject in need thereof, the method comprising: administering to said subject an effective amount of flaxseed, its bioactive ingredient, degradant or a metabolite thereof.
- Administration to said subjects encompasses administration prior to, during and after exposure to damaging exposure to chemical carcinogens and toxicants both natural and synthetic.
- the time prior, during and post could be seconds, minutes, hours, days, weeks, months or even years.
- the bioactive ingredient encompasses secoisolaricirecinol diglucoside (SDG), secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or a combination thereof.
- SDG secoisolaricirecinol diglucoside
- SECO secoisolariciresinol
- ED enterodiol
- EL enterolactone
- the invention relates to a method for protecting a biomolecule, from carcinogen damage resulting from accidental exposure to chemical carcinogens and toxicants both natural and synthetic in a subject in need thereof, the method comprising: administering to said subject an effective amount of flaxseed, its bioactive ingredient, degradant or a metabolite thereof.
- the invention relates to a method for protecting a biomolecule (such as genetic material like a nucleic acid, a protein or a lipid), a cell, or a tissue, from damage from chemical carcinogens and toxicants both natural and synthetic resulting in lung cancer or mesothelioma.
- a biomolecule such as genetic material like a nucleic acid, a protein or a lipid
- the invention in another aspect, relates to a method for protecting a biomolecule (such as genetic material like a nucleic acid, a protein or a lipid), a cell, or a tissue, from carcinogen damage, the method comprising: contacting said biomolecule, cell, or tissue with an effective amount of a bioactive ingredient.
- a biomolecule such as genetic material like a nucleic acid, a protein or a lipid
- a cell such as a protein or a lipid
- a tissue comprising: contacting said biomolecule, cell, or tissue with an effective amount of a bioactive ingredient.
- Contact with said biomolecule, cell, or tissue encompasses contact prior to, during and after exposure to damaging exposure to chemical carcinogens and toxicants both natural and synthetic.
- the time prior, during and post could be seconds, minutes, hours, days, weeks, months or even years.
- the bioactive ingredient encompasses secoisolaricirecinol diglucoside (SDG), secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or a combination thereof.
- SDG secoisolaricirecinol diglucoside
- SECO secoisolariciresinol
- ED enterodiol
- EL enterolactone
- the invention relates to a method for treating or preventing carcinogen-induced damage, malignant transformation or cancer development in subject who has been or will be exposed to one or more carcinogens from carcinogen-induced cancer, the method comprising: administering to said subject an effective amount of at least one bioactive ingredient, wherein said bioactive ingredient comprises secoisolaricirecinol diglucoside (SDG), secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or a combination thereof.
- SDG secoisolaricirecinol diglucoside
- SECO secoisolariciresinol
- ED enterodiol
- EL enterolactone
- the invention in another aspect, relates to a method for protecting a subject exposed to one or more carcinogens from a carcinogen-induced cancer, the method comprising: administering to said subject an effective amount of flaxseed, its bioactive ingredient, or a metabolite thereof.
- the invention relates to a method for protecting a biomolecule (such as genetic material like a nucleic acid, a protein or a lipid), a cell, or a tissue, from damage by hypochlorite ions in a subject in need thereof, the method comprising: administering to said subject an effective amount of flaxseed, its bioactive ingredient, degradant or a metabolite thereof.
- Administration to said subjects encompasses administration prior to, during and after exposure to damaging exposure to chemical carcinogens and toxicants both natural and synthetic.
- the time prior, during and post could be seconds, minutes, hours, days, weeks, months or even years.
- the bioactive ingredient encompasses secoisolaricirecinol diglucoside (SDG), secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or a combination thereof.
- SDG secoisolaricirecinol diglucoside
- SECO secoisolariciresinol
- ED enterodiol
- EL enterolactone
- the invention relates to a method for treating or preventing hypochlorite ion-induced damage in a subject who has been or will be exposed to hypochlorite ions, the method comprising: administering to said subject an effective amount of at least one bioactive ingredient, wherein said bioactive ingredient comprises secoisolaricirecinol diglucoside (SDG), secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or a combination thereof.
- SDG secoisolaricirecinol diglucoside
- SECO secoisolariciresinol
- ED enterodiol
- EL enterolactone
- the invention in another aspect, relates to a method for protecting a biomolecule (such as genetic material like a nucleic acid, a protein or a lipid), a cell, or a tissue, from damage by hypochlorite ions, the method comprising: contacting said biomolecule, cell, or tissue exposed to or to be exposed to hypochlorite ions with an effective amount of a bioactive ingredient.
- Contact with said biomolecule, cell, or tissue encompasses contact prior to, during and after exposure to damaging exposure to chemical carcinogens and toxicants both natural and synthetic.
- the time prior, during and post could be seconds, minutes, hours, days, weeks, months or even years.
- the bioactive ingredient encompasses secoisolaricirecinol diglucoside (SDG), secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or a combination thereof.
- SDG secoisolaricirecinol diglucoside
- SECO secoisolariciresinol
- ED enterodiol
- EL enterolactone
- the invention relates to a composition for use in one of the foregoing methods.
- FIG. 1 Effect of increasing doses of ⁇ radiation on Plasmid (pBR322) DNA relaxation.
- Super coiled (SC) represents the compact form.
- the open coiled (OC) form represents the relaxed or the damaged form of the plasmid.
- (A) - The super coiled (SC) form is the lower prominent band (at 3,000 bps) while the open coiled (OC) form is the upper prominent band.
- Figure 2 Effect of increasing concentration of synthetic SDG (S,S), SDG (R,R) and commercial SDG on ⁇ radiation-induced Plasmid (pBR322) DNA relaxation. All samples were exposed to a ⁇ radiation dose of 25 Gray. SDGs concentrations were 25, 50, 100 and 250 ⁇ .
- Figures (A), (D), and (G) - representative agarose gel scans of plasmid DNA after exposure to 25 Gy in the presence of 25, 50, 100 and 250 ⁇ SDG (S,S), SDG (R,R) and SDG (commercial) are shown.
- Figures (B), (E) and (H) - SC and OC forms are presented as percent of total plasmid DNA. For each condition, all samples were run in duplicates. The data is presented as mean + standard deviation. P ⁇ 0.05 was considered significant. * and # show the significant difference as compared to untreated SC and OC forms, respectively. ** and ## show significant differences as compared to samples exposed to 25 Gy without SDGs.
- SDGs - dependent inhibition of plasmid DNA relaxation is shown. EC50 values were determined from the quadratic equations presented under the curves.
- Figure 3 Effect of increasing doses of ⁇ radiation on calf thymus DNA fragmentation.
- DNA exposed to ⁇ -radiation generates fragments of small molecular weights which move faster than the higher molecular wt. DNA.
- Determining the density of the low molecular wt DNA fragments ( ⁇ 6,000 bps) as compared to the high molecular wt. DNA (>6,000 bps) reflects the extent of radiation-induced damage.
- (A) Lane 1 - 1 kb DNA standard ladder, lanes 2 and 3 - untreated calf thymus DNA, lanes 4 and 5 - DNA exposed to 25 Gy, and lanes 6 and 7 - DNA exposed to 50 Gy.
- Figure 4 Effect of increasing concentration of synthetic SDG (S,S), SDG (R,R) and commercial SDG on ⁇ radiation-induced calf thymus DNA fragmentation. All samples were exposed to a ⁇ radiation dose of 50 Gray. SDGs concentrations were 25, 50, 100 and 250 ⁇ .
- Figures (A), (C), and (E)_- representative agarose gel scans of calf thymus DNA after exposure to 50 Gy in the presence of 25, 50, 100 and 250 ⁇ SDG (S,S), SDG (R,R) and SDG (commercial) are shown.
- Figures (B), (D) and (F) - High and Low molecular wt DNA forms are presented as percent of total DNA. For each condition, all samples were run in duplicates. The data is presented as mean + standard deviation. P ⁇ 0.05 was considered significant. * shows the significant difference as compared to untreated DNA. # shows the significant difference as compared to samples exposed to 50 Gy without SDGs.
- Figure 5 Effect of very low concentrations of synthetic SDG (5", 5"), SDG (R,R) and commercial SDG on ⁇ radiation-induced calf thymus DNA fragmentation. All samples were exposed to a ⁇ radiation dose of 50 Gy. SDGs concentrations were 0.5, 1.0, 5.0 and 10 ⁇ .
- Figures (A), (C), and (E) - representative agarose gel scans of calf thymus DNA after exposure to 50 Gy in the presence of 0.5, 1.0, 5.0 and 10 ⁇ SDG (S,S), SDG (R,R) and SDG (commercial) are shown.
- Figures (B), (D) and (F) - High and Low molecular wt DNA forms are presented as percent of total DNA. For each condition, all samples were run in duplicates. The data is presented as mean + standard deviation. P ⁇ 0.05 was considered significant. * shows the significant difference as compared to untreated DNA. # shows the significant difference as compared to samples exposed to 50 Gy without SDGs.
- Figure 6 Effect of SDG, SECO, ED and EL on ⁇ radiation-induced calf thymus DNA fragmentation. All samples were exposed to a ⁇ radiation dose of 50 Gy. SDG, SECO, ED and EL were used at 10 ⁇ concentration.
- Figure 7 Effect of SDG treatment on radiation dose response of murine primary lung cells.
- A Epithelial Cells;
- B Endothelial Cells and
- C WT Fibroblasts.
- Cells were treated with different concentrations of SDG for 6 h prior to gamma irradiation (0, 2, 4, 6, 8 Gy) and incubated. All visible colonies were counted on 12- 14th day and surviving fraction was normalized against control values. Data is represented as mean +SEM. ** p ⁇ 0.001 * p ⁇ 0.01, #p ⁇ 0.05 for irradiated cells VS 50 ⁇ SDG pre-treated irradiated cells.
- Figure 8 Evaluation of the radiation-induced DNA single strand breaks (SSB) in lung cells using the alkaline comet assay.
- A Kinetics evaluation of DNA damage in 2- Gy gamma irradiated primary lung cells (epithelial, endothelial and WT Fibroblasts); At least 100-150 cells were counted for each treatment. DNA damage was assessed by calculating the "tail moment" for each cell (the product of amount of DNA in tail, times the tail length). * p ⁇ 0.001 for non-irradiated controls VS their respective irradiated cells;
- B Effect of SDG (50 ⁇ ) treatment (0, 2, 4, 6 hours prior to irradiation) on irradiated primary lung cells.
- FIG. 9 Fluorescent evaluation of the induction of ⁇ - ⁇ 2 ⁇ foci in irradiated murine primary lung cells viz. epithelial cells, endothelial cells and WT fibroblasts. Cells were treated with SDG (50 ⁇ ) for 6h and gamma-irradiated (2 Gy). At desired time interval, cells were fixed in 4% paraformaldehyde, washed, probed with ⁇ - ⁇ 2 ⁇ antibody and nuclei was counterstained using DAPI.
- Figure 10 Representative panels of immunofluorescence visualization of ⁇ - H2AX foci (green) in murine lung epithelial cells.
- Cells were pre-treated with SDG for 6h, gamma-irradiated (2 Gy) and incubated further for 30 min.
- Cells were fixed in 4% paraformaldehyde and probed with ⁇ - ⁇ 2 ⁇ antibody.
- DNA was counterstained with DAPI (blue). Images were acquired using a fluorescence microscope.
- FIG 11 Flow cytometric (FACS) confirmation of the induction of ⁇ - ⁇ 2 ⁇ foci in irradiated murine primary lung cells.
- Primary lung cells viz. epithelial cells (A), endothelial cells (B) and WT Fibroblasts (C) were treated with SDG (50 uM) for 6h and gamma-irradiated (2 Gy). At desired time interval, cells were processed for FACS analysis. Data was quantified using Summit software and is represented as mean +SEM. *p ⁇ 0.05, ** p ⁇ 0.01 for irradiated cells VS SDG pre-treated irradiated cells.
- Figure 12 Evaluation of the radiation-induced apoptotic death in primary lung cells. Quantitative evaluation of the effect of SDG (50 mM) pre-treatment (6 h) on irradiated primary lung cells viz. epithelial cells (A), endothelial cells (B) and WT Fibroblasts (C). Cells were fixed, stained with DAPI and visualized for morphological analysis under fluorescence microscope. For each treatment, at least 500 cells were counted from 5 different fields and percentage of apoptotic cells was calculated. Experiment was done twice. Data is represented as mean +SEM. *p ⁇ 0.05, ** p ⁇ 0.005 for irradiated cells VS. SDG pre-treated irradiated cells.
- FIG. 13 Evaluation of the effect SDG treatment on regulators of apoptosis in lung epithelial cells.
- Murine primary lung cells epithelial cells
- SDG 50 ⁇
- Cells were harvested at 6, 24, and 48 hours post-irradiation.
- Total RNA was isolated from epithelial cells at desired time interval and evaluated by quantitative real time RT-PCR analysis for Bax and Bcl-2 gene expression (A and B). Analysis was performed in triplicate and gene expression was normalized to 18S ribosomal RNA.
- Bax and Bcl-2 protein levels were assessed by western blot analysis; representative images (C) and densitometry analysis with normalization to ⁇ -actin. (D and E). Data is represented as mean + SEM. *p ⁇ 0.05, **p ⁇ 0.01 for IR-exposed cells compared to either SDG treated cells or SDG + IR treated cells.
- Figure 14 Effect of SDG on radiation induced increases in levels of active caspase-3 and cleaved PARP.
- Murine primary lung cells epidermal cells
- SDG 50 ⁇
- Cells were harvested at 6, 24, and 48 hours post-radiation.
- Cleaved caspase-3 and cleaved PARP protein levels were assessed by western blot analysis.
- A Representative images and (B and C) densitometry analysis with normalization to ⁇ -actin. Analysis was performed in duplicate and data is represented as mean +SEM. *p ⁇ 0.05, **p ⁇ 0.01 for IR-exposed cells compared to SDG treated cells.
- Figure 15 SDG Scavenges Hypochlorite Ions.
- Figure 15A shows the CIO " dependent increase in APF and HPF fluorescence.
- Figure 15B shows scavenging of CIO " by SDG.
- Figure 15C shows the scavenging effect of synthetic SDG diastereomers SDG(S, S) and SDG (R, R). All samples were run in duplicates. The data are presented as mean + standard error. P ⁇ 0.05 was considered significant. * shows the significant difference as compared to untreated control.
- Figure 16 SDG Scavenges ⁇ - Radiation-induced Generation of Hypochlorite.
- Figures 16A and B show ⁇ - radiation-induced increase APF and HPF fluorescence.
- FIGS 16C and D show the effect of SDG on generation of hypochlorite, at increasing doses of radiation in Phosphate buffered saline (PBS) with either APF or HPF (see Figure
- Figures 16E, F and G show ⁇ -radiation-induced chlorination of Taurine.
- Figure 16E shows hypochlorite-dependent chlorination of taurine.
- Figure 16F shows the taurine chloramine as absorbance for all experimental conditions.
- Figure 16G shows the hypochlorite concentration under various conditions as in Figure 16F.
- Figures 16A-E all samples were run in duplicates whereas for Figures 16F and G, all samples were run in quadruplets. The data are presented as mean + standard error. P ⁇ 0.05 was considered significant. * shows the significant difference as compared to the untreated controls.
- Figure 17 Hypochlorite-induced Calf thymus DNA Damage.
- Figures 17A and C show representative agarose gels scans of calf thymus DNA after exposure to HOC1.
- Figures 17B and D show high and low molecular wt DNA fragments as percent of total DNA.
- Figures 17E and F show the effect of SDG on hypochlorite-induced damage to plasmid DNA.
- Figure 17E shows a representative agarose gel of plasmid DNA after exposure to HOC1.
- Figure 17F shows SC and OC forms presented as percent of total plasmid DNA.
- Figure 18 Effect of SDG (Pre-and Post-treatment) on Hypochlorite-induced Modification of 2-Aminopurine (2-AP).
- Figure 18A shows the representative spectra for all the conditions.
- Figure 18B shows the fluorescence at 374 nm under different conditions as in Figure 18 A.
- Figure 18C shows the % protection by SDG. For each condition, all samples were run in duplicates. The data are presented as mean + standard error. P ⁇ 0.05 was considered significant. * and # show the significant difference as compared to untreated 2-AP control and treated, respectively.
- Figure 19 SDG prevents ⁇ - radiation-induced modification of 2-aminopurine (2-AP).
- Figure 19A shows the representative spectra for all the conditions.
- Figure 19B shows the fluorescence at 374 nm. For each condition, all samples were run in duplicates. The data are presented as mean + standard error. P ⁇ 0.05 was considered significant. * and # show the significant difference as compared to untreated 2-AP control and treated, respectively.
- Figure 20 Proposed mechanism of SDG action in DNA protection from nucleobase chlorination.
- Figure 21 Mechanism of chemoprevention by flaxseed and its lignans. SDG mitigates lung tumorigenesis by tobacco and other environmental carcinogens by inhibiting the multi-step carcinogenesis process.
- the lignan SDG has chemopreventive activity through modulation of the Nrf2-regulated Phase ⁇ detoxification pathway, and perhaps other mechanisms, in both animal models.
- FIG. 22 SDG decreases oxidative DNA damage induced by benzo-alpha- pyrene in cells.
- SDG (10 ⁇ ) was added to human epithelial cells (A549) that were exposed to 25 ⁇ of the tobacco and environmental carcinogen benzo-alpha-pyrene (BaP) and oxidative damage to DNA was detected using mass spectrometry as indicated by the presence of 8-oxo-7,8-dihydroguanine (8-oxo-dGuo).
- SDG decreased DNA damage at 3 and 6 hours post carcinogen exposure.
- Figure 23 The carcinogen benzo-alpha-pyrene induced ROS in cells. Exposure of murine epithelial cells to BaP induces damaging reactive oxygen species (ROS) as detected by a redox- sensitive fluorescence dye. As early as 2 hours, post exposure to the carcinogen, a robust increase of fluorescence intensity indicates ROS generation in cells.
- ROS reactive oxygen species
- FIG. 24 SDG prevents ROS generation from carcinogen exposure.
- Mouse epithelial cells were exposed to 10 or 20 ⁇ BaP and an increasing concentration of SDG (0, 0.1, 0.5, 1, 5 ⁇ SDG) and ROS was detected 2 hours later (as determined appropriate in Figure 23).
- SDG scavenged harmful ROS to negligible levels.
- Figure 25 SDG prevents genotoxic stress in human epithelial cells exposed to BaP. Exposure of cells to a potent carcinogen such as BaP, induces genotoxic stress as indicated by increased levels of p53 protein. This is mitigated dose-dependently by the presence of SDG, at 5, 10, 25 and 50 ⁇ concentration.
- Figure 26 SDG prevents oxidative DNA damages in human epithelial cells exposed to BaP. Exposure of cells to a potent carcinogen such as BaP, induces oxidative DNA damage as indicated by increased levels of gamma-H2AX, a marker for double- stranded DNA breaks. This is mitigated dose-dependently by the presence of SDG, at 5, 10, 25, 50 and 100 ⁇ concentration.
- FIG. 27 SDG prevents DNA adduct formation in human epithelial cells exposed to BaP. Exposure of cells to a potent carcinogen such as BaP, induces the formation of DNA adducts. DNA adducts are pieces of DNA covalently linked to a carcinogen and is directly linked to the development of malignancy. The DNA adduct levels is decreased by the presence of SDG or its metabolites ED and EL given alone or in combination.
- Figure 28 Mouse model of chemical carcinogen-induced lung tumors. Mice (A/J strain) are given 4 injections intraperitoneally of the tobacco and environmental carcinogen BaP (once weekly) at 1 mg/Kg dose. Mice are initiated on flaxseed or lignan diet at the time of exposure. Mice are evaluated at various times post exposure to determine tumor burden, mouse weight, and overall health profile.
- Figure 29 Flaxseed decreases tumor burden in mice: Gross pathological profile of murine lungs exposed to carcinogen. Representative clinical images of murine lungs several months post BaP exposure and dietary flaxseed administration.
- Figure 30 Flaxseed decreases tumor burden in mice: Histopathological profile of murine lungs exposed to carcinogen. Representative H&E-strained lung sections of murine lungs several months post BaP exposure and dietary flaxseed administration. Nodules indicated by the arrows from mice fed control diet (top panels) or flaxseed (lower panels) appear smaller in the flaxseed- fed mice. Each panel represents a different animal.
- FIG. 31 Flaxseed decreases tumor burden in mice: Quantitative assessment of tumor burden. Histological murine lung sections were evaluated morphologically using image analysis software for overall tumor area (A) and nodule size (B). There was a significant decrease in the area of the lung occupied by tumor in the mice fed a flaxseed diet (p ⁇ 0.03). Similarly, there was a trend for smaller tumor nodule size.
- Figure 32 Flaxseed decreases tumor burden in mice: Quantitative assessment of tumor burden. Histological murine lung sections were evaluated morphologically using Image analysis software for overall number of tumor nodules per lung (A) and % tumor invading the lung (B). There was a trend for less tumor nodules per lung (A) and less tumor invading with flaxseed supplementation (B)
- Figure 33 Flaxseed supplementation prevents wasting effects from lung cancer induced by BaP. Animal weight was measured longitudinally for 200 days post BaP exposure. Mice fed a flaxseed diet, exposed to BaP showed higher weight than those exposed to BaP on control diet.
- Figure 35 Mammalian lignan metabolites are detectable in blood 4 days after daily ingestion of Flaxseed (FS) and Flaxseed Lignan Component (FLC)-supplemented diets. Specifically, Enterodiol (ED) and Enterolactone (EL) can be detected using liquid chromatography, tandem mass spectrometry (LC/MS/MS). Diets were designed to deliver comparable lignan levels, reflected in the detectable lignan metabolite levels in the 2 diets.
- FS Flaxseed
- FLC Flaxseed Lignan Component
- ED Enterodiol
- EL Enterolactone
- Diets were designed to deliver comparable lignan levels, reflected in the detectable lignan metabolite levels in the 2 diets.
- Figure 36 Flaxseed (FS) and Flaxseed Lignan Component (FLC) given prior to asbestos exposure, blunted abdominal inflammation induced by ip crocidolite asbestos injection as evidenced by the numbers of macrophages (MF), neutrophils (PMN) and lymphocytes (Ly). Specifically, FS and FLC significantly decreased macrophage influx in the abdomen. *p ⁇ 0.05
- FIG 37 Mice were initiated on FS and FLC diets and exposed to asbestos 24 hours later. Cytokines levels (TNFa and IL- ⁇ ) in plasma (B, D) and in abdomen, (A, C) were determined using ELISA 3 days post exposure according to the experimental scheme in Figure 34. Both diets indicated a trend towards preventing secretion of proinflammatory cytokines in the abdomen and the systemic circulation induced by asbestos exposure.
- Figure 38 Inflammatory cells also trended lower with the diet (A) while TNFa (b) and IL- ⁇ (C) cytokine levels induced by 400 and 800 mg crocidolite asbestos were significantly blunted by FLC added in the diet 1 day post asbestos exposure (*p ⁇ 0.05). *p designates significance as compared to control diet exposed to asbestos.
- Figure 39 Plasma concentration of SDG and metabolites following oral gavage of variable SDG concentrations in mice.
- Figure 40 Antioxidant enzyme gene expression levels in lung following oral gavage of variable doses of SDG in mice.
- Figure 41 Kinetics of SDG levels in plasma (A) and lung tissues (B) following oral gavage of 100 mg/Kg SDG in mice and corresponding levels of AOE gene expression (C).
- Figure 43 Clinical study design for Example 7.
- Figure 44 Western Blot showing that feeding 10% FS increases HO-1 and NQO-1 in mouse nasal epithelium.
- Figure 45 Kinetics of HO-1 gene expression in human buccal epithelial cells after 40g FS diet. (*P ⁇ 0.05 from 0 day).
- Figure 46 Kinetics of urinary IsoP levels in one patient on FS.
- Figure 47 Kinetics of urinary 8-oxo-dGuo levels in Normal and Lung transplant patients on FS.
- Figure 48 SDG or flaxseed diets are hypothesized to decrease asbestos induced ROS/inflammation.
- Figure 49 Experimental plan (schematic) of asbestos exposure of cells to detect inflammatory cytokine secretion and nitrosative/oxidative stress:
- Figure 50 SDG blunts asbestos-induced ROS secretion by human mesothelial cells in vitro.
- FIG 51 Evaluation of Asbestos-Induced Oxidative Stress (ROS release) in Culture RAW Macrophages: Asbestos-induced ROS was generated shortly post asbestos exposure and continued for the duration of the observation period.
- Figure 52 SDG given to macrophages several hours post exposure to asbestos decreases oxidative stress.
- Figure 53 SDG given to macrophages several hours post exposure to asbestos decreases nitrosative stress.
- Figure 54 SDG given to macrophages several hours post exposure to asbestos decreases inflammatory cytokine secretion (IL- ⁇ ).
- Figure 55 SDG given to macrophages several hours post exposure to asbestos decreases inflammatory cytokine secretion (TNF-a).
- Figure 56 Testing SDG in Asbestos-Induced Mesothelioma using two mouse models: Using at least 2 models of mice genetically predisposed to develop mesothelioma after asbestos exposure, we will: Evaluate the acute effects of Flaxseed and SDG on a single dose of asbestos in mice; test whether Flaxseed and SDG inhibits the development of tumors in genetic models of accelerated, asbestos induced MM.
- Figure 58 Experimental Plan of asbestos exposure of NF2 mice and flaxseed/SDG lignan formulation evaluation:
- Figure 59 Kinetics of abdominal inflammation in NF2 mice post asbestos exposure: Inflammatory cell influx peaked by 3 days and tapered off by 9 days post asbestos exposure. Therefore, 3 days was selected as the time point to evaluate inflammation in all subsequent experiments.
- Figure 60 Flaxseed and its SDG-rich lignan component blunted asbestos- induced inflammation (younger mice): Total white blood cells (A) decreased with FS or FLC addition in the diet, albeit not significantly. However, when looking at cell differentials, and macrophage levels in particular, levels were significantly blunted by both flaxseed and the SDG-lignan diet (B).
- FIG. 61 Flaxseed and its SDG-rich lignan component blunted asbestos- induced inflammation (older mice): Older mice exposed to abdominal asbestos (A) are more sensitive to asbestos by presenting with approx. 3,000,000 WBC /mL of abdominal lavage fluid as compared to just 300,000 cells/mL (10-fold higher). Results indicated that the inflammatory cells Neutrophils (B) and Macrophages (C) were both significantly higher in older than in younger mice.
- FIG. 62 Flaxseed lignan extract enriched in SDG (given in diet formulation) blunts asbestos inflammation in older mice: Male NF2 (129SV) (+/-) mice were injected (intraperitoneal) with 400 ⁇ g of asbestos on Day 0. Mice were initiated on the test diets (0% FS or 10% FLC) the week prior to asbestos exposure (Day-7) and sacrificed on Day 3 post-asbestos exposure. Abdominal lavage (AL) was performed with 5 mL lx PBS (1 ml of belly lavage fluid was centrifuged and the supernatant was frozen). Plasma was collected at frozen at -80°. Cells were evaluated in lavage fluid and showed that total WBC and neutrophils, macrophages and eosinophils were all significantly decreased by the SDG-rich diet.
- FIG. 63 Flaxseed lignan extract enriched in SDG (given in diet formulation) blunts asbestos inflammatory cytokine secretion and nitrosative stress in older mice: Male NF2 (129SVX+/-) mice were injected (intraperitoneal) with 400 ⁇ g of asbestos on Day 0. Mice were initiated on test diets (0% FS or 10% FLC) the week prior to asbestos exposure (Day 7) and sacrificed on Day 3 post-asbestos exposure. Abdominal lavage (AL) was performed with 5 mL lx PBS (1 mL of belly lavage fluid was centrifuged and the supernatant frozen). Plasma was collected at frozen at -80°. Levels of cytokines ILi and TNFa as well as nitrites were significantly blunted by the SDG-rich diet.
- A Abdominal lavage
- the bioactive ingredient comprises secoisolariciresinol diglucoside (SDG), secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, isomers (including stereoisomers) thereof, or a combination thereof.
- SDG secoisolariciresinol diglucoside
- SECO secoisolariciresinol
- ED enterodiol
- EL enterolactone
- flaxseed, its bioactive ingredients, and/or its degradants or metabolites are effective in protecting biomolecules, cells, and tissues from radiation damage, hypochlorite ion-induced damage, carcinogen- induced damage and malignancy. Accordingly, the inventors have found that flaxseed, its bioactive ingredients, or its metabolites can be used for protecting biomolecules, cells, and tissues from radiation damage, hypochlorite ion-induced damage, carcinogen damage and cancer development.
- Subjects in need of radioprotection or radiation mitigation according to methods provided herein are subjects who will, are, or have been exposed to potentially deleterious amounts of radiation. It will be understood that such exposure may be a single exposure, periodic exposure, sporadic exposure or ongoing exposure to the radiation. It is also understood that such radiation exposure includes accidental exposure, incidental or intentional exposure.
- Examples of subjects who may be in need of radioprotection or radiation mitigation according to the methods of the present invention include but are not limited to, patients who are exposed to radiation as part of therapeutic regimen (e.g. , cancer patients who require radiation therapy), subjects who are exposed to radiation for to diagnose a disease or condition (e.g. , subjects receiving dental or bone X-rays, patients receiving PET scans, CT scans and the like).
- Examples of subjects who may be in need of radioprotection or radiation mitigation according to the methods of the present invention also include those who may be exposed to radiation as a result of their profession or life style choices (e.g.
- Subjects in need of chemoprevention according to methods provided herein are subjects who will, are, or have been exposed to potentially deleterious amounts of carcinogens or other toxicants. It will be understood that such exposure may be a single exposure, periodic exposure, sporadic exposure or ongoing exposure to one or combination of several synthetic or naturally occurring carcinogens or other toxicants, such as chemical warfare agents. It is also understood that such exposure includes accidental exposure, incidental or intentional exposure. It will also be understood that such exposure may be direct exposure or indirect exposure. For example, indirect exposure to hypochlorite ions may be the result of direct exposure to ionizing radiation.
- Examples of subjects who may be in need of chemoprevention according to the methods of the present invention include but are not limited to those who may be exposed to carcinogens or other toxicants as a result of their profession or life style choices (e.g. , workers in the oil industry; toll booth attendants exposed to automobile exhaust particles; laboratory technicians and other workers).
- Other subjects who may be in need of chemoprevention according to the methods of the present invention include those who are accidentally exposed to carcinogens, such as leaks or spills of carcinogens in the drinking water or the air (asbestos, polyaromatic hydrocarbons).
- Also contemplated are those exposed to carcinogens as a result of a habit (smokers).
- Additional subjects encompassed are those who are exposed to a terrorist's act to disperse carcinogen and other cancer promoting materials, such as chemical warfare agents.
- the invention relates to a method for protecting a biomolecule (such as genetic material like a nucleic acid, a protein or a lipid), a cell, or a tissue, from radiation damage in a subject in need thereof, the method comprising: administering to said subject an effective amount of flaxseed, its bioactive ingredient, degradant or a metabolite thereof.
- Administration to said subjects encompasses administration prior to, during and after exposure to damaging radiation exposure.
- the time prior, during and post could be seconds, minutes, hours, days, weeks, months or even years.
- the bioactive ingredient encompasses secoisolaricirecinol diglucoside (SDG), secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or a combination thereof.
- SDG secoisolaricirecinol diglucoside
- SECO secoisolariciresinol
- ED enterodiol
- EL enterolactone
- the invention in another aspect, relates to a method for treating or preventing radiation damage in a subject who has been or will be exposed to radiation, the method comprising: administering to said subject an effective amount of at least one bioactive ingredient, wherein said bioactive ingredient comprises secoisolaricirecinol diglucoside (SDG), secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or a combination thereof.
- SDG secoisolaricirecinol diglucoside
- SECO secoisolariciresinol
- ED enterodiol
- EL enterolactone
- the invention relates to a method for protecting biomolecule (such as genetic material like a nucleic acid, a protein or a lipid), a cell, or a tissue, from radiation damage resulting from accidental radiation exposure in a subject in need thereof, the method comprising: administering to said subject an effective amount of flaxseed, its bioactive ingredient, degradant or a metabolite thereof.
- biomolecule such as genetic material like a nucleic acid, a protein or a lipid
- cell or a tissue
- the invention relates to a method for protecting biomolecule (such as genetic material like a nucleic acid, a protein or a lipid), a cell, or a tissue, from radiation damage resulting in aging.
- biomolecule such as genetic material like a nucleic acid, a protein or a lipid
- the invention relates to a method for protecting a biomolecule (such as genetic material like a nucleic acid, a protein or a lipid), a cell, or a tissue from damage resulting from exposure to chemical carcinogens and toxicants, including chemical warfare agents, both natural and synthetic.
- a biomolecule such as genetic material like a nucleic acid, a protein or a lipid
- a cell or a tissue from damage resulting from exposure to chemical carcinogens and toxicants, including chemical warfare agents, both natural and synthetic.
- the invention relates to a method for protecting a biomolecule (such as genetic material like a nucleic acid, a protein or a lipid), a cell, or a tissue, from radiation damage resulting from radiation therapy for cancer treatment in a subject in need thereof, the method comprising: administering to said subject an effective amount of flaxseed, its bioactive ingredient, degradant or a metabolite thereof.
- a biomolecule such as genetic material like a nucleic acid, a protein or a lipid
- a cell such as a protein or a lipid
- a tissue comprising: administering to said subject an effective amount of flaxseed, its bioactive ingredient, degradant or a metabolite thereof.
- the invention relates to a method for protecting a biomolecule (such as genetic material like a nucleic acid, a protein or a lipid), a cell, or a tissue from radiation damage in a subject in need thereof, the method comprising: administering to said subject an effective amount of at least one bioactive ingredient, wherein said bioactive ingredient comprises secoisolariciresinol diglucoside (SDG), secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or a combination thereof.
- the radiation damage results from accidental radiation exposure.
- the radiation damage results from radiation therapy for cancer (e.g. , lung cancer) treatment.
- the invention relates to a method for preventing radiation induced damage to a biomolecule (such as genetic material like a nucleic acid, a protein or a lipid), a cell, or a tissue, in a subject in need thereof, the method comprising: administering to said subject an effective amount of flaxseed, its bioactive ingredient, or a metabolite thereof.
- a biomolecule such as genetic material like a nucleic acid, a protein or a lipid
- the invention relates to a method for preventing radiation induced damage to a biomolecule (such as genetic material like a nucleic acid, a protein or a lipid), a cell, or a tissue, in a subject in need thereof, the method comprising: administering to said subject an effective amount of at least one bioactive ingredient, wherein said bioactive ingredient comprises secoisolariciresinol diglucoside (SDG), secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or a combination thereof.
- SDG secoisolariciresinol diglucoside
- SECO secoisolariciresinol
- ED enterodiol
- EL enterolactone
- the invention relates to a method for protecting a biomolecule (such as genetic material like a nucleic acid, a protein or a lipid), a cell, or a tissue, from radiation damage in a cell, the method comprising contacting said cell with an effective amount of at least one bioactive ingredient, wherein said bioactive ingredient comprises secoisolariciresinol diglucoside (SDG), secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or a combination thereof.
- SDG secoisolariciresinol diglucoside
- SECO secoisolariciresinol
- ED enterodiol
- EL enterolactone
- the invention in another aspect, relates to a method for protecting a biomolecule (such as genetic material like a nucleic acid, a protein or a lipid), a cell, or a tissue, from carcinogen damage in a subject in need thereof, the method comprising: administering to said subject an effective amount of flaxseed, its bioactive ingredient, degradant or a metabolite thereof.
- Administration to said subjects encompasses administration prior to, during and after exposure to damaging exposure to chemical carcinogens and toxicants both natural and synthetic.
- the time prior, during and post could be seconds, minutes, hours, days, weeks, months or even years.
- the bioactive ingredient encompasses secoisolaricirecinol diglucoside (SDG), secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or a combination thereof.
- SDG secoisolaricirecinol diglucoside
- SECO secoisolariciresinol
- ED enterodiol
- EL enterolactone
- the invention relates to a method for protecting a biomolecule, from carcinogen damage resulting from accidental exposure to chemical carcinogens and toxicants both natural and synthetic in a subject in need thereof, the method comprising: administering to said subject an effective amount of flaxseed, its bioactive ingredient, degradant or a metabolite thereof.
- the invention relates to a method for protecting a biomolecule (such as genetic material like a nucleic acid, a protein or a lipid), a cell, or a tissue, from damage from chemical carcinogens and toxicants both natural and synthetic resulting in lung cancer or mesothelioma.
- a biomolecule such as genetic material like a nucleic acid, a protein or a lipid
- the invention relates to a method for protecting a biomolecule (such as genetic material like a nucleic acid, a protein or a lipid), a cell, or a tissue, from carcinogen damage, the method comprising: contacting said biomolecule, cell, or tissue with an effective amount of a bioactive ingredient.
- a biomolecule such as genetic material like a nucleic acid, a protein or a lipid
- a cell or a tissue
- the method comprising: contacting said biomolecule, cell, or tissue with an effective amount of a bioactive ingredient.
- Contact with said biomolecule, cell, or tissue encompasses contact prior to, during and after exposure to damaging exposure to chemical carcinogens and toxicants both natural and synthetic.
- the time prior, during and post could be seconds, minutes, hours, days, weeks, months or even years.
- the bioactive ingredient encompasses secoisolaricirecinol diglucoside (SDG), secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or a combination thereof.
- SDG secoisolaricirecinol diglucoside
- SECO secoisolariciresinol
- ED enterodiol
- EL enterolactone
- the invention relates to a method for treating or preventing carcinogen-induced damage, malignant transformation or cancer development in subject who has been or will be exposed to one or more carcinogens from carcinogen-induced cancer, the method comprising: administering to said subject an effective amount of at least one bioactive ingredient, wherein said bioactive ingredient comprises secoisolaricirecinol diglucoside (SDG), secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or a combination thereof.
- SDG secoisolaricirecinol diglucoside
- SECO secoisolariciresinol
- ED enterodiol
- EL enterolactone
- the invention relates to a method for protecting a subject exposed to one or more carcinogens from a carcinogen-induced cancer, the method comprising: administering to said subject an effective amount of flaxseed, its bioactive ingredient, or a metabolite thereof.
- the invention relates to a method for protecting a biomolecule (such as genetic material like a nucleic acid, a protein or a lipid), a cell, or a tissue, from damage by hypochlorite ions in a subject in need thereof, the method comprising: administering to said subject an effective amount of flaxseed, its bioactive ingredient, degradant or a metabolite thereof.
- Administration to said subjects encompasses administration prior to, during and after exposure to damaging exposure to chemical carcinogens and toxicants both natural and synthetic.
- the time prior, during and post could be seconds, minutes, hours, days, weeks, months or even years.
- the bioactive ingredient encompasses secoisolaricirecinol diglucoside (SDG), secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or a combination thereof.
- SDG secoisolaricirecinol diglucoside
- SECO secoisolariciresinol
- ED enterodiol
- EL enterolactone
- the invention relates to a method for treating or preventing hypochlorite ion-induced damage in a subject who has been or will be exposed to hypochlorite ions, the method comprising: administering to said subject an effective amount of at least one bioactive ingredient, wherein said bioactive ingredient comprises secoisolaricirecinol diglucoside (SDG), secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or a combination thereof.
- SDG secoisolaricirecinol diglucoside
- SECO secoisolariciresinol
- ED enterodiol
- EL enterolactone
- the invention in another aspect, relates to a method for protecting a biomolecule (such as genetic material like a nucleic acid, a protein or a lipid), a cell, or a tissue, from damage by hypochlorite ions, the method comprising: contacting said biomolecule, cell, or tissue exposed to or to be exposed to hypochlorite ions with an effective amount of a bioactive ingredient.
- a biomolecule such as genetic material like a nucleic acid, a protein or a lipid
- a cell, or a tissue from damage by hypochlorite ions
- the method comprising: contacting said biomolecule, cell, or tissue exposed to or to be exposed to hypochlorite ions with an effective amount of a bioactive ingredient.
- Contact with said biomolecule, cell, or tissue encompasses contact prior to, during and after exposure to damaging exposure to chemical carcinogens and toxicants both natural and synthetic.
- the time prior, during and post could be seconds, minutes, hours, days, weeks, months or even years.
- the bioactive ingredient encompasses secoisolaricirecinol diglucoside (SDG), secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or a combination thereof.
- SDG secoisolaricirecinol diglucoside
- SECO secoisolariciresinol
- ED enterodiol
- EL enterolactone
- the invention relates to a composition for use in one of the foregoing methods.
- Flaxseed, its bioactive ingredients, and its metabolites are known in the art and described in U.S. Patent Publication Nos. 2010/0239696; 2011/0300247; and 2014/0308379; and in International Patent Publication No. WO2014/200964, each of which is incorporated by reference herein in its entirety.
- the primary lignan found in flaxseed is 2,3-bis (3-methoxy-4-hydroxybenzyl) butane- 1,4-diol (secoisolariciresinol or SECO), which is stored as the conjugate secoisolariciresinol diglucoside (SDG) in its native state in the plant.
- SDG secoisolariciresinol diglucoside
- ED enterodiol
- EL enterolactone
- a “degradant” is a product of the breakdown of a molecule, such as SDG, into smaller molecules.
- a "metabolite” is a substance produced by metabolism or by a metabolic process.
- a metabolite of SDG is EL or ED.
- a metabolite may be a chemically synthesized equivalent of a natural metabolite.
- An "analog” is a compound whose structure is related to that of another compound.
- the analog may be a synthetic analog.
- An "ingredient” or “component” is an element or a constituent in a mixture or compound.
- a "product” is a substance resulting from a chemical reaction.
- An "extract” is a preparation containing an active principle or concentrated essence of a material, for example, from flaxseed.
- “Pharmaceutical composition” refers to an effective amount of an active ingredient, e.g. , (S,S)-SOG (R,R)-SOG, meso-SOG, SDG, SECO, EL, ED and analogs thereof, together with a pharmaceutically acceptable carrier or diluent.
- a “therapeutically effective amount” refers to that amount which provides a therapeutic effect for a given condition and administration regimen.
- compositions described herein may include a "therapeutically effective amount.”
- a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
- a therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual.
- a therapeutically effective amount is also one in which toxic or detrimental effects of the molecule are outweighed by the therapeutically beneficial effects.
- the phrase "pharmaceutically acceptable” refers to those compounds, materials, compositions, carriers, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
- “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes an excipient that is acceptable for veterinary use as well as human pharmaceutical use.
- a “pharmaceutically acceptable excipient” as used herein includes both one and more than one such excipient.
- compositions can be administered to a subject by any suitable method known to a person skilled in the art, such as orally, parenterally, transmucosally, transdermally, intramuscularly, intravenously, intra-dermally, subcutaneously, intra-peritonealy, intra-ventricularly, intra-cranially, intra-vaginally, intratumorally, or bucally.
- Controlled release may also be used by embedding the active ingredient in an appropriate polymer which may then be inserted subcutaneously, intratumorally, bucally, as a patch on the skin, or vaginally. Coating a medical device with the active ingredient is also covered.
- the pharmaceutical compositions are administered orally, and are thus formulated in a form suitable for oral administration, i.e., as a solid or a liquid preparation.
- Suitable solid oral formulations include tablets, capsules, pills, granules, pellets and the like.
- Suitable liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils and the like.
- the active ingredient is formulated in a capsule.
- the compositions of the present invention comprise, in addition to the active compound and the inert carrier or diluent, drying agent, in addition to other excipients as well as a gelatin capsule.
- the pharmaceutical compositions are administered by intravenous, intra-arterial, or intra-muscular injection of a liquid preparation.
- the pharmaceutical composition is a liquid preparation formulated for oral administration.
- the pharmaceutical composition is a liquid preparation formulated for intravaginal administration. Suitable liquid formulations include solutions, suspensions, dispersions, emulsions, oils and the like.
- the pharmaceutical compositions are administered intravenously and are thus formulated in a form suitable for intravenous administration.
- the pharmaceutical compositions are administered intra-arterially and are thus formulated in a form suitable for intra-arterial administration.
- the pharmaceutical compositions are administered intra-muscularly and are thus formulated in a form suitable for intra-muscular administration. In some embodiments, the pharmaceutical compositions are administered intra-bucally and are thus formulated in a form suitable for buccal administration.
- the pharmaceutical compositions are administered topically to body surfaces and are thus formulated in a form suitable for topical administration.
- suitable topical formulations include gels, ointments, creams, lotions, drops, controlled release polymers and the like.
- the flaxseed, its bioactive ingredient, or a metabolite thereof is prepared and applied as a solution, suspension, or emulsion in a physiologically acceptable diluent with or without a pharmaceutical carrier.
- the pharmaceutical compositions provided herein are controlled-release compositions, i.e. compositions in which the flaxseed, its bioactive ingredient, or a metabolite thereof is released over a period of time after administration.
- Controlled- or sustained-release compositions include formulation in lipophilic depots (e.g. fatty acids, waxes, oils).
- the composition is an immediate- release composition, i.e. a composition in which all the flaxseed, its bioactive ingredient, or a metabolite thereof is released immediately after administration.
- compositions for use in the methods provided herein are administered at a therapeutic dose once per day. In some embodiments, the compositions are administered once every two days, twice a week, once a week, or once every two weeks.
- (S,S)-SDG (R,R)-SDG, (S,R)-SDG (R,S)-SDG, meso-SDG, SECO, EL, ED or an analog thereof may be administered at a dose of 0.1 ng/kg to 500 mg/kg.
- the treatment with (S.S)-SDG (R,R)-SDG, (S,R)-SDG (R,S)-SDG, meso-SDG, SDG, SECO, EL, ED or an analog thereof is a single administration to several days, months, years, or indefinitely.
- treating may refer to either therapeutic treatment or prophylactic or preventative measures, wherein the object is to prevent or lessen the targeted pathologic condition or disorder as described herein, or both. Therefore, compositions for use in the methods provided herein may be administered to a subject before the exposure, e.g. , to radiation, a carcinogen, a toxicant, or hypochlorite ions. In some cases, compositions for use in the methods provided herein may be administered to a subject after the exposure. Thus treating a condition as described herein may refer to preventing, inhibiting, or suppressing the condition in a subject.
- the terms “treat” and “treatment” refer to therapeutic treatment, as well prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change associated with a disease or condition.
- Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of the extent of a disease or condition, stabilization of a disease or condition (i.e. , where the disease or condition does not worsen), delay or slowing of the progression of a disease or condition, amelioration or palliation of the disease or condition, and remission (whether partial or total) of the disease or condition, whether detectable or undetectable.
- Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
- Those in need of treatment include those already having been exposed, e.g. , to radiation, a carcinogen, a toxicant, or hypochlorite ions, as well as those prone to being exposed or those expecting to be exposed.
- subjects in need of treatment and the methods and compositions described herein may include, but are not limited to, subjects with lung diseases and disorders, such as asthma, cancer, COPD, and mesothelioma.
- suitable subjects may include subjects with disorders and conditions associated with aging, such as cardiovascular disorders and conditions, sagging skin and central nervous system (CNS) diseases (e.g. , Alzheimer's dementia).
- suitable subjects may include skin disorders and conditions (e.g. , psoriasis), as well as subjects with cosmetic skin conditions (e.g. , wrinkles and age spots).
- suitable subjects may include subjects with gastrointestinal disorders and conditions, such as IBD and chron' s disease.
- suitable subjects may include subjects with cardiovascular disorders and conditions. In some embodiments, suitable subjects may include subjects with melanoma. In some embodiments, suitable subjects may include subjects with ocular diseases, such as macular degeneration. In some embodiments, suitable subjects may include subjects with cancer, such as breast cancer, prostate cancer and uterine cancer. In some embodiments, suitable subjects includesubjects with cognitive impairment and other cognitive disorders.
- the term "subject” includes mammals, e.g. , humans, companion animals (e.g. , dogs, cats, birds, and the like), farm animals (e.g., cows, sheep, pigs, horses, fowl, and the like) and laboratory animals (e.g. , rats, mice, guinea pigs, birds, and the like).
- the subject may include dogs, cats, pigs, cows, sheep, goats, horses, buffalo, ostriches, guinea pigs, rats, mice, birds (e.g. , parakeets) and other wild, domesticated or commercially useful animals (e.g., chicken, geese, turkeys, fish).
- the term "subject” does not exclude an individual that is normal in all respects.
- the term “subject” includes, but is not limited to, a human in need of therapy for, or susceptible to, a condition or its sequelae.
- the electromagnetic wave has a very small wavelength ( ⁇ 0.005 nm) and thus has high energy which is capable of ionizing molecules and atoms.
- ionizing radiation generates hydroxyl radicals ( ⁇ ) by water radiolysis. These hydroxyl radicals ( ⁇ ) are the predominant source of ionizing radiation-induced damage to cellular components including lipids, proteins and genomic DNA.
- the hydroxyl radicals ( ⁇ ) produced by ⁇ - radiation result in single-strand and double-strand breaks in DNA.
- the ( OH) radicals damage DNA by abstracting H-atoms from the deoxyribose and purine as well as pyrimidine bases or add to the double bonds of the bases. These reactions result in DNA strand breaks.
- SDG secoisolariciresinol diglucoside
- SDG has been shown in many studies by Christofidou-Solomidou et al , in addition to others, to be a potent antioxidant agent and a potent free radical scavenger.
- the synthetic SDG enantiomers have been shown to possess strong antioxidant and free radical, scavenging characteristics (Mishra et ah, Bioorganic & Medicinal Chemistry Letters 2013, (19):5325-5328).
- the radioprotective properties of the synthesized SDG enantiomers (S.Sj-SDG and ⁇ R,R)-$DG as they compare to the commercial SDG were investigated and evaluated.
- the radioprotective characteristics of the three compounds were assessed using the plasmid DNA relaxation assay by determining the ability of the SDGs to prevent the super coil to open coil plasmid DNA (pBR322) conversion following the exposure of the plasmid to ⁇ - irradiation as well as by evaluating inhibition of genomic DNA fragmentation following the exposure of the DNA to ⁇ -irradiation.
- SDG is metabolized by intestinal bacteria to produce secoisolariciresinol (SECO), enterodiol (ED) and enterolactone (EL). Therefore, the effect of these metabolites of SDG on ⁇ -irradiation-induced fragmentation of genomic DNA was also evaluated.
- Plasmid DNA (pBR322), ethidium bromide, ultrapure 10X TAE buffer and 1 kb plus DNA ladder were purchased from Invitrogen (Life Technologies, Carlsbad, CA). Agarose (ultrapure) and calf thymus DNA were purchased from Sigma-Aldrich (St. Louis, MO). Secoisolariciresinol Diglucoside (SDG) (commercial), Secoisolariciresinol (SECO), enterodiol (ED) and enterolactone (EL) were purchased from Chromadex (Irvine, CA). Synthesis of Secoisolariciresinol Diglucoside (SDG)
- Plasmid DNA pBR322
- calf thymus DNA samples with or without varying concentrations of SDG (R,R), SDG (S,S) and SDG (commercial) were exposed to ⁇ -radiation with a Mark 1 cesium (Cs-137) irradiator (J.L. Shepherd, San Fernando, CA) at a dose rate of 1.7 Gy/min in phosphate buffered saline, pH 7.4 (PBS).
- Cs-137 Mark 1 cesium irradiator
- the gel was stained with ethidium bromide (0.5 ⁇ g/ml) for 40 min, washed for 20 min and then visualized on a UV trans-illuminator (Bio-Rad, Hercules, CA).
- the captured gel images were scanned and the density of the open coiled (OC) and super coiled (SC) plasmid DNA bands determined by Gel-doc image analyzer program.
- the density of the SC and OC plasmid DNA was expressed as % of the total density (OC + SC).
- the gel was stained with ethidium bromide (0.5 ⁇ g/ml) for 40 min, washed for 20 min and then visualized on a UV trans-illuminator (Bio-Rad, Hercules, CA).
- the captured gel images were scanned and the density of the calf thymus DNA fragments was determined by Gel-Pro image analyzer program (Media Cybernetics, Silver Spring, MD).
- the density of the low mol. wt ( ⁇ 6,000 bps) and the high mol. wt (>6,000 bps) fragments of calf thymus DNA was expressed as % of the total density (low mol wt. + high mol. wt.).
- the radioprotective potential of synthetic SDG (R,R), SDG (S,S) and SDG (commercial) was determined using plasmid DNA (pBR322).
- the radioprotection assay used in this study is based on the principle that plasmid DNA following exposure to ⁇ - radiation moves slower than the unexposed plasmid DNA. This is simply due to the fact that the super coiled plasmid DNA moves faster in the agarose gel due to its compact size. By comparison, the radiation-induced nicks in the plasmid DNA unravel super coil resulting in a relatively lager size plasmid which moves with a slower rate in the gel. Therefore, determining the density of the open coiled as compared to super coiled plasmid DNA reflects the extent of radiation-induced damage.
- the % of OC increased from 31.26+2.50% to 49.08+2.31% 61.62+3.73% and 67.33+4.24% (p ⁇ 0.05), in 0, 10, 25 and 50 Gy, respectively.
- a radiation dose of 25 Gy was selected for the subsequent experiments to determine the radioprotective characteristics of the different SDGs.
- Plasmid DNA was exposed to the selected dose of 25 Gy gamma radiation (see Figure 1) and the % inhibition of DNA damage (SC to OC formation) was determined for each of the SDG agents (synthetic and commercial) at various concentrations (25-250 ⁇ ).
- FIG. 2A A representative gel blot of plasmid DNA after exposure to 25 Gy in the presence of 25, 50, 100 and 250 ⁇ SDG (S,S) is shown in Figure 2A and semiquantitative densitometric analysis is shown in Figure 2B while % inhibition as compared to control is shown in Figure 2C.
- SDG SDG
- the EC 50 value can be determined for each agent (i.e, the effective concentration (EC) needed to prevent 50% of plasmid relaxation at 25 Gy) which for SDG (5,5) is 141.77 ⁇ .
- This value for preventing plasmid DNA relaxation is comparable to the EC 50 value for scavenging DPPH free radicals.
- FIG. 4A shows a representative DNA gel of calf thymus DNA after exposure to 50 Gy in the presence of 25, 50, 100 and 250 ⁇ SDG (5,5).
- SDG SDG
- Figure 4B shows the distribution of high and low mol. wt size DNA forms in presence of various concentrations of SDG (S,S) in Figure 4B.
- results presented in Figures 4C-D and 4E-F show the radioprotective properties of synthetic SDG (R,R) and SDG (commercial), respectively. These results demonstrate the radioprotective characteristic of synthetic SDG (R,R) and (S, S) enantiomers using calf thymus genomic DNA.
- SDG (S,S) and SDG (R,R) completely prevented the radiation induced generation of low mol. wt. fragments of calf thymus DNA demonstrating strong radioprotective characteristics of synthetic SDG (S,S) and SDG (R,R) enantiomers.
- the mammalian lignan metabolites of SDG, SECO, ED and EL showed equally potent DNA-protective properties.
- Flavonoids possess strong antioxidant activity; specifically, such polyphenols possess free radical-scavenging activities, and are known to be more effective antioxidants in vitro than vitamins E and C. Dietary and medicinal plants possessing antioxidant properties are also known to prevent many human diseases associated with oxidative stress and are useful radioprotectors. Antioxidants, including vitamins and minerals, suppressed the levels of clastogenic factors in Chernobyl workers many years after radiation exposure. We have been investigating the role of whole grain dietary flaxseed, a grain rich in lignan polyphenols, as well as of flaxseed lignan formulations enriched in SDG, in radiation- induced damage using a mouse model of thoracic radiation damage.
- flaxseed ameliorated the radiation-induced inflammation and oxidative stress in mice when administered both prior to and after radiation exposure.
- irradiated mice fed diets containing only the lignan component of flaxseed, enriched in the lignan biphenol SDG also showed significantly improved hemodynamic measurements and survival while also improving lung inflammation and oxidative tissue damage.
- ROS reactive oxygen species
- SDG Reactive oxygen species result in cellular damage by oxidative modification of cellular membrane lipids, proteins and the genomic DNA.
- a number of studies have shown that extracted, purified or synthetic flaxseed SDG is a potent anti-oxidant in vitro as well as in vivo. Therefore, SDG as an antioxidant has therapeutic potential under various experimental and disease conditions including radiation-induced tissue damage in patients undergoing radiation therapy.
- SDG Secoisolariciresinol
- SDG flaxseed lignan secoisolariciresinol
- the antioxidant properties of the SDG molecule have been previously demonstrated. We have shown that natural, commercially available SDG has potent free- radical scavenging properties in cells exposed to gamma radiation.
- the antioxidant and free radical scavenging characteristics of these synthetic SDG (R,R) and SDG (5,5) enantiomers were investigated and have been demonstrated to possess strong reducing power, high metal-ion chelating potential, and high free radical scavenging activity for hydroxyl, peroxyl and DPPH radicals. These characteristics of the synthetic SDG (R,R) and SDG (5,5) indicate that these molecules show strong potential for modulating cellular redox state, decreasing metal-ion concentration, and scavenging oxygen free radicals.
- SDG as an antioxidant and free radical scavenger can also function as a DNA radioprotector and radiation mitigator.
- ROS reactive oxygen species
- antioxidants can be ROS scavengers that interfere with free radical chain reactions, it is possible to protect cellular DNA from radiation-induced oxidative stress by supplementation with antioxidants.
- a number of synthetic and natural antioxidant compounds have been studied for their radioprotective efficacy. However most of them exhibit inherent toxicity and side effects at their effective concentrations, or have short shelf life and low bioavailability. Hence the search for effective and non-toxic radioprotectors has led to investigations into dietary antioxidants and nutraceuticals.
- dietary flaxseed a flaxseed supplementation in preclinical murine models of oxidative lung damage such as hyperoxia, acid aspiration injury, and ischemia/reperfusion injury.
- oxidative lung damage such as hyperoxia, acid aspiration injury, and ischemia/reperfusion injury.
- FS dietary flaxseed
- dietary flaxseed ameliorated the adverse effects of thoracic radiation when given both prior to exposure as well as post-exposure.
- dietary flaxseed decreased radiation-induced oxidative lung tissue damage, decreased lung inflammation and prevented pulmonary fibrosis.
- SDG Secoisolariciresinol diglucoside
- ED enterodiol
- EL enterolactone
- FS lignans are protective against diverse cancer types as summarized in a recent review on the health effects of SDG by Adolphe et al (Br J Nutr 2010, 103:929) and reported to reduce melanoma metastasis in animals.
- This study was performed to determine the radioprotective ability of FS lignan SDG and to explore the possible mechanisms responsible for its action.
- the first aim of this study was to evaluate role of SDG on radiation-induced clonogenic death in primary murine lung cells, specifically in epithelial, endothelial cells and fibroblasts. Since radiation-induced reproductive death of cells is directly related to cellular DNA damage, we assessed whether SDG can protect cells from radiation-induced DNA strand breaks by using alkaline comet assay (SSBs) and formation of ⁇ - ⁇ 2 ⁇ foci (DSBs). Furthermore, we examined the effect of SDG pre-treatment in preventing murine primary lung cells from IR-induced cell death.
- SSBs alkaline comet assay
- DSBs formation of ⁇ - ⁇ 2 ⁇ foci
- SDG Secoisolariciresinol Diglucoside
- PBS Phosphate buffered saline
- BSA Bovine serum albumin
- DMEM Dulbecco's modified Eagle's medium
- trypsin bovine serum albumin
- BSA bovine serum albumin
- EDTA ethylenediamine tetra acetic acid
- DAPI 4,6diamidino 2-phenyl indole
- FBS Fetal bovine serum
- Collagenase Triton-X 100 and Dispase were purchased from Sigma-Aldrich, St. Louis, MO, USA.
- Fibroblasts and endothelial cells were isolated from C57/bl6 mouse. For fibroblast isolation, mouse lungs were harvested, minced, and incubated with dispase (2 mg/ml) for 45 minutes. Pieces were plated out and fibroblasts were cultured as described previously and used between passages 3 and 10.
- Pulmonary microvascular endothelial cells (PMVEC) were isolated from murine lungs as described previously. Briefly, freshly harvested mouse lungs were treated with collagenase followed by isolation of cells by adherence to magnetic beads coated with mAb to platelet endothelial cell adhesion molecule (PEC AM).
- Epithelial cells (CIO) cells were originally derived from a normal BALB/c mouse lung explant and are non-tumorigenic, contact-inhibited, and have alveolar type 2 cell features at early passage.
- Exponentially growing cells were plated as single cells and incubated overnight. Cells were treated with various doses of the lignan SDG (10-50 ⁇ ) 6 h prior to irradiation (2, 4, 6 and 8 Gy). Lignan dose was selected based on animal studies to be within the physiological levels reached in the blood circulation when 10% Flaxseed is ingested. Cells were irradiated with a Mark 1 cesium (Cs-137) irradiator (J.L. Shepherd, San Fernando, CA) at a dose rate of 1.7 Gy/min. Colonies were stained and counted 10 to 15 days after irradiation and surviving fraction was calculated.
- Cs-137 Mark 1 cesium
- Colonies were stained and counted 10 to 15 days after irradiation and surviving fraction was calculated.
- PBST PBS + 0.1% TritonX-100 containing 5% goat serum, 1% BSA.
- Cells were incubated with ⁇ - ⁇ 2 ⁇ primary antibody (1:200) overnight at 4°C followed by washing with PBST (3x5 min) and incubation with secondary antibody (Alexa fluor® 488, Invitrogen, CA, USA) for 1 hr at RT. Nuclei were counterstained with DAPI and visualized under fluorescence microscope.
- Apoptotic cells are morphologically characterized by condensation of nucleus and cytoplasm, membrane blebbing, cell shrinkage, and breakdown of nuclear DNA, first in large segments and subsequently in nucleosomal fragments and finally formation of well-enclosed apoptotic bodies. Percentage of cells undergoing apoptosis was determined microscopically from the slides used for micronuclei detection (cytogenetic damage). At least, 500 cells were counted for each experiment (experiment done twice) and percent apoptotic cells were determined as follows:
- N a is the number of cells with apoptotic bodies and N t is the total number of cells analyzed.
- Apoptosis was determined in mouse lung epithelial cells by levels of Bax (an apoptosis promoter), Bcl-2 (an apoptosis inhibitor), cleaved caspase-3, and cleaved poly (adenosine diphosphate-ribose) polymerase (PARP) seen using immunoblotting. Briefly, cells were lysed in PBS containing protease inhibitors. Immunoblot analysis of cell lysates was then performed using 10 well SDS 12% NuPAGE gel (Invitrogen, Carlsbad California). Electrophoresis was performed at 200V for 1 hour.
- Bax an apoptosis promoter
- Bcl-2 an apoptosis inhibitor
- cleaved caspase-3 cleaved caspase-3
- PARP cleaved poly (adenosine diphosphate-ribose) polymerase
- Protein levels of Bax, Bcl-2, cleaved caspase-3, and cleaved PARP were detected using manufacturer recommended dilutions (Cell Signaling Technology, Danvers, MA) using rabbit anti-mouse monoclonal antibody against BAX and Bcl-2, and rabbit anti-mouse cleaved caspase-3 (Aspl75), monoclonal antibody and a rabbit polyclonal anti-cleaved PARP (214/215) cleavage site specific antibody.
- the membrane was washed five times and then incubated in secondary antibody conjugated to horseradish peroxidase for 45 minutes at room temperature.
- Membranes were developed using Western Lighting Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Boston, MA) and quantified by densitometry scanning of specific bands (20 kDa for Bax, 26 kDa for Bcl-2, 17/19 kDa for cleaved caspase-3, and 89 kDa for cleaved PARP) that were adjusted for loading using ⁇ -actin expression level detected by specific secondary antibody (Sigma, St. Louis, MO).
- Results are expressed as mean + SEM. Survival curve for clonogenic assay was prepared using KaleidaGraph software (4.0). Statistical differences among groups were determined using one-way analysis of variance (ANOVA). When statistically significant differences were found (p ⁇ 0.05) individual comparisons were made using the Bonferoni/Dunn test (Statview 4.0).
- SDG treatment increases colony forming ability of irradiated primary lung cells
- the clonogenic survival assay has been used widely to determine cellular reproductive death after a cell undergoes any genotoxic stress after exposure to environmental and pharmaceutical carcinogens, ionizing radiation etc.
- SDG 10-50 ⁇
- results show that SDG (10-50 ⁇ ) alone did not elicit any adverse effect on the colony forming ability of all the three cell types as compared to their respective untreated control cells (100%) ( Figure 1A-1C).
- SDG prevents formation of DNA SSBs in irradiated primary lung cells
- SDG pre-treatment (6 hours prior IR) significantly decreased the induction of ⁇ - ⁇ 2 ⁇ , as the number of ⁇ - ⁇ 2 ⁇ positive cells decreased to 22.7%+ 2.17, 21.92%+2.88 and 22.1%+1.9 in irradiated epithelial cells, endothelial cells and fibroblasts, respectively (p ⁇ 0.005 for epithelial, and p ⁇ 0.05 for endothelial and fibroblasts).
- the ability of SDG to protect cells from the formation of ⁇ - ⁇ 2 ⁇ foci appeared to be independent of cell type as pre-treatment of SDG protected all three types of lung cells from radiation-induced DNA strand breaks.
- Figure 10 depicts a representative fluorescence photomicrograph of microscopic analysis of ⁇ - ⁇ 2 ⁇ positive cells in primary lung epithelial cells.
- SDG treatment prevents primary lung cells from IR-induced apoptotic death
- SDG treatment modifies the expression of regulators of apoptosis in murine primary lung epithelial cells
- FS lignan SDG can protect murine primary lung cells against radiation-induced oxidative DNA damage and apoptotic death.
- SDG pre-treatment not only improved IR-induced cytotoxicity as measured by clonogenic survival, but also decreased the induction of DNA strand breaks (DSBS and SSBS) and cell death in lung cells.
- expression of genes implicated in the regulation of apoptosis was also altered by SDG treatment.
- IR-induced cell death is a classical marker of cellular radiosensitivity, which is characterized by loss in clonogenic survival. Similarly, we also noticed a radiation dose dependent loss in clonogenicity in all three murine lung cells which was attenuated significantly by SDG pre-treatment (Figure 7).
- Cellular DNA is the primary target of IR-induced damage in the cell. ROS generated during exposure induces an array of changes in cellular DNA ranging from mutations, base lesions, cross-linking, SSBs and DSBs. Damage in DNA cannot be replaced and thus must be repaired and if it remains unrepaired, the cell may resort to induction of apoptosis or necrosis.
- the comet assay is a sensitive technique for the detection of DNA damage/repair at the cellular level and has been used widely to investigate DNA strand breaks.
- Polyphenols have the ability to protect normal tissue or cells from damaging effects of radiation by reducing ROS mediated oxidative DNA damage.
- SDG standard alkaline comet assay
- DSBs While SSBs are easily repaired by the cell, DSBs are more difficult for cells to repair and are more likely to result in mutagenesis, hence DSBs represent mostly the lethal cellular event.
- H2AX molecules become phosphorylated ( ⁇ - ⁇ 2 ⁇ ) along megabase-long chromatin domains for each DNA double-strand break introduced by irradiation and ⁇ - ⁇ 2 ⁇ loss or de-phosphorylation correlates time-wise with DNA repair.
- SDG protected cellular DNA from IR-induced DSBs in all three types of cells tested. Our results are in agreement with some other studies which also show that polyphenols like Resveratrol and green tea catechin protect cells from IR-induced DNA strand breaks. However, in the absence of radiation, SDG did not exert any toxic effect in either of these cells. As oxidative DNA damage is considered to be a precursor to many cancers, a reduction in such damage by SDG acting as antioxidant may lead to reduced risk of cancer.
- SDG Secoisolariciresinol Diglucoside
- Hypochlorous acid (HOC1), a potent oxidant, is produced by neutrophils by activated myeloperoxidase which catalyzes the reaction between physiologically present chloride ions and hydrogen peroxide (H2O2). Activated neutrophils produce H2O2 and superoxide anion 0 2 ⁇ ⁇ .
- HOC1 kills microorganisms by oxidative damage. However, excessive production is known to cause damage to tissues.
- Hypochlorite modifies adenine nucleotides resulting in formation of chloramines that appears to be a major mechanism of neutrophil-mediated toxicity.
- HOC1 and its conjugated base CIO have been shown to oxidize amino acids, peptides, proteins and lipids and to chlorinate bases in cellular DNA and RNA.
- the reaction of HOCI/CIO " results in modification of both purine and pyrimidine nucleotides at the endocyclic -NH groups of guanine and thymine as well as the exocyclic NH2 groups of guanine, adenine and cytosine derivatives resulting in the formation of chloramines such as (RNHC1) and RR'NCl.
- the primary modified bases were found to be 5-chlorocytosine, 8-chloroadenine and 8-chloroguanine in DNA and RNA of SKM-1 cells.
- ⁇ -radiation is capable of ionizing atoms and molecules.
- ionizing radiation generates hydroxyl radicals ( ⁇ ) which are believed to be the source of ionizing radiation-induced damage to cellular components including lipids, proteins and DNA.
- CI " hydroxyl radicals
- these highly unstable radicals can be scavenged by CI " ions which are present in the physiological medium at very high concentrations. This leads to generation of reactive chlorine-containing intermediates, among which relatively stable CIO " is the radiation-derived toxicant.
- CIO " and other active chlorine derivatives of oxidative nature are formed as products of radiolysis; they can contribute to suppression of physiological functions of organisms. Therefore, we propose that radiation-induced DNA or protein damage is mediated, in part, by radiation-generated CIO " .
- ROS indicator probes APF and HPF, plasmid DNA (pBR322), and 1 kb plus DNA ladder were from Invitrogen (Life Technologies, Carlsbad, CA).
- Calf thymus or plasmid DNA was incubated with hypochlorite for 2 hrs at 37 °C. DNA samples were subjected to agarose (1%) gel electrophoresis and analyzed.
- SDG a known lignan antioxidant and free radical scavenger, detoxified hypochlorite ions generated in physiological solutions by chemical means as well as by radiation
- SDG protected genomic DNA as well as plasmid DNA from hypochlorite-induced damage
- iii The mechanism of SDG defense (protection or recovery from hypochlorite-induced DNA damage), involves scavenging hypochlorite and regeneration of amino (-NH) groups on nucleobases from chloramines (-NC1);
- SDG action is equally effective in protecting DNA from hypochlorite-induced damage when added prior to or post-exposure, i.e., it can act as a protector and/or mitigator of
- HOCl is produced by myeloperoxidase of activated neutrophils using hydrogen peroxide generated by NADPH oxidase and chloride ions as substrates. HOCl can chlorinate and oxidize nucleobases. Since HOCl can chlorinate nucleobases, this might cause genotoxicity. Chlorinated nucleosides have been identified and linked to inflammation and cancer.
- DNA from radiation-induced damage 1) by scavenging hypochlorite ions that cause chlorination and oxidation of nucleobases; 2) by scavenging * OH free radicals that produce hypochlorite by reacting with chloride ions.
- * OH was drastically decreased in the presence of chloride ion in phosphate buffered saline (PBS) as well as in saline alone (preliminary experiments).
- hypochlorite and hypochlorite-induced damage to cellular components including DNA would be a predominant mechanism of radiation damage; 3) by associating with DNA base pairs as several flavonoids such as lutiolin, kempferol and quercetin; 4) by blocking abstraction of protons or addition of * OH on the purine and pyrimidine bases especially at C5, C6 and C8, and at the deoxyribose sites. These mechanisms have been proposed for protection from free radical-induced DNA damage; 5) lastly, by reduction of chloroamines formed, thus, regenerating internal and external amino groups in nucleic acids.
- SDG as a scavenger of hypochlorite ions as well as being an antioxidant and free radical scavenger and protector of nucleobases from hypochlorite-induced chlorination can function as a DNA radioprotector and as a radiation mitigator.
- hypochlorite exists in solution as a mixture of hypochlorite anion (CIO " ), hypochlorous acid (HCIO) and free chlorine (Cl 2 ) in pH-dependent amounts. At physiological pH, CIO " and HCIO are the predominant molecules. Unlike strong, single- electron oxidants such as * OH, hypochlorite is a two-electron oxidant, less reactive and more selective than * OH. Hypochlorite can chlorinate electron-rich aromatic rings and NH- compounds. It oxidizes primary and secondary alcohols as well as benzyl methylene groups and tertiary methine groups, and phenols. The first step of the above reactions is chlorination followed by hydrolysis/HCl elimination.
- SDG contains all the above reaction sites except amino groups that make the SDG molecule a potent hypochlorite scavenger.
- Scheme 1 Figure 20 we proposed a novel mechanism of DNA protection by SDG using either a 1 -electron or a 2-electron reduction of a nucleobase.
- hypochlorite CIO "
- hypochlorite ions are known to modify DNA bases by chlorination/oxidation and then subsequently resulting in DNA damage
- our findings show SDG may be useful as a radioprotector of normal tissue damage associated with radiotherapy for cancer treatment or accidental exposure to radiation.
- Flaxseed, SDG and lignan derivatives mitigate lung tumorigenesis by tobacco and other environmental carcinogens by inhibiting the multi-step carcinogenesis process (Figure 21).
- experiments are provided indicating that the lignan SDG has chemopreventive activity through modulation of the Nrf2-regulated Phase ⁇ detoxification pathway, and perhaps other mechanisms, in animal models.
- the protective effects of SDG are mediated by the direct ROS scavenging and/or indirect antioxidant/anti-inflammatory properties, and decrease of carcinogen toxicity and DNA damage.
- SDG decreases oxidative DNA damage induced by carcinogens in cells
- SDG (10 ⁇ ) was added to human epithelial cells (A549) that were exposed to 25 ⁇ of BaP and oxidative damage to DNA was detected using mass spectrometry as indicated by the presence of 8-oxo-7,8-dihydroguanine (8-oxo-dGuo).
- SDG prevents ROS generation from carcinogen exposure
- mouse epithelial cells were exposed to 10 or 20 ⁇ BaP and an increasing concentration of SDG (0, 0.1, 0.5, 1, 5 ⁇ SDG) and ROS was detected 2 hours later.
- SDG scavenged harmful ROS to negligible levels ( Figure 24).
- SDG prevents genotoxic stress in human epithelial cells exposed to BaP. Exposure of cells to a potent carcinogen such as BaP, induces genotoxic stress as indicated by increased levels of p53 protein ( Figure 25). This is mitigated dose- dependently by the presence of SDG, at 5, 10, 25 and 50 ⁇ concentration.
- SDG also prevents oxidative DNA damages in human epithelial cells exposed to BaP. Exposure of cells to a potent carcinogen such as BaP, induces oxidative DNA damage as indicated by increased levels of gamma-H2AX, a marker for double-stranded DNA breaks (Figure 26). This is mitigated dose-dependently by the presence of SDG, at 5, 10, 25, 50 and 100 ⁇ concentration. Furthermore, SDG prevents DNA adduct formation in human epithelial cells exposed to BaP. Exposure of cells to a potent carcinogen such as BaP, induces the formation of DNA adducts (Figure 27). DNA adducts are pieces of DNA covalently linked to a carcinogen and is directly linked to the development of malignancy. The DNA adduct levels is decreased by the presence of SDG or its metabolites ED and EL given alone or in combination.
- Flaxseed and its lignans provides protection in a mouse model of chemical carcinogen-induced lung tumors.
- Mice (A/J strain) are given 4 injections intraperitoneally of the tobacco and environmental carcinogen BaP (once weekly) at 1 mg/Kg dose (Figure 28).
- Mice are initiated on flaxseed or lignan diet at the time of exposure. Mice are evaluated at various times post exposure to determine tumor burden, mouse weight, and overall health profile.
- Flaxseed decreases tumor burden in mice:
- Figure 29 presents representative clinical images of murine lungs several months post BaP exposure and dietary flaxseed administration.
- Figure 30 presents representative H&E-strained lung sections of murine lungs several months post BaP exposure and dietary flaxseed administration. Nodules indicated by the arrows from mice fed control diet (top panels) or flaxseed (lower panels) appear smaller in the flaxseed-fed mice. Each panel represents a different animal.
- Histological murine lung sections were evaluated morphologically using image analysis software for overall tumor area and nodule size ( Figures 31A and B). There was a significant decrease in the area of the lung occupied by tumor in the mice fed a flaxseed diet (p ⁇ 0.03). Similarly, there was a trend for smaller tumor nodule size. Histological murine lung sections were also evaluated morphologically for overall number of tumor nodules per lung ( Figure 32A) and % tumor invading the lung ( Figure 32B). There was a trend for less tumor nodules per lung (A) and less tumor invading with flaxseed supplementation (B).
- Flaxseed and its lignans protect cells and tissues from asbestos-induced damage Introduction
- MM Malignant mesothelioma
- FS Whole grain Flaxseed
- FS and its lignan component, (FLC) enriched in the lignan secoisolariciresinol diglucoside (SDG) was given in rodent chow.
- Mice (Nf2 + mut ;Cdkn2a + mut ) were, placed a day later (+1) or prior (-1) to a single ip bolus of 400mg of crocidolite asbestos on 10% FS or 10% FLC supplemented diets and evaluated 3 days later for abdominal inflammation and proinflammatory cytokine release (see Figure 34).
- the NF2 mouse strain was selected as it develops an accelerated form of MM when exposed to asbestos.
- LC/MS/MS tandem mass spectrometry
- systemic levels i.e., plasma
- flaxseed lignan metabolites such as the mammalian lignans Enterolactone (EL) and Enterodiol (ED) were evaluated to ensure that FS was effectively metabolized by the gut flora of this mouse strain and that levels were comparable to those in other mouse models.
- EL mammalian lignans Enterolactone
- ED Enterodiol
- Flaxseed can thus, be used as a dietary agent in the chemoprevention of malignant mesothelioma.
- Gene expression levels of HOI, NQOl and GST increased 2-3 fold with as little as 5 mg SDG/Kg and reached an average of 6-fold increase over baseline with 100 mg/Kg SDG. Gene levels are supported by lower but yet significant increase in protein levels in lung tissues.
- Plasma and lung tissue concentrations of SDG reached levels of 0.8 and 12.6 ⁇ at 30 minutes post-administration, respectively.
- robust induction of representative AOE gene expression levels (HO-1, NQOl, GST) were significantly elevated (p ⁇ 0.05) over baseline ( Figure 41C).
- the observed gene expression increase correlated with an increase in protein levels determined by western blotting (not shown).
- SDG at the same dose given twice daily for 7 days showed neither intolerance nor toxicity.
- Nrf2 knockout mice Data from lung injury models show that FS and FS-derived SDG in diets can upregulate Nrf2 and Nrf2-actived genes and proteins in lungs.
- Nrf2 knockout mice the hypothesis that activation of this pathway is an important mechanism of the chemopreventive effects of SDG is tested and it is determined if SDG has activity in both the initiation and promotion phases of carcinogenesis. Effects of SDG in Nrf2-deficient mice.
- SDG dosing studies and kinetics determined that SDG should be given for at least a few days, as this was determined to be the minimum time required for the diets to reach steady state in tissues, followed by 4 weekly injections of 1 mg/mouse Benzo[a]pyrene (BaP) given i.p. Both 50 and 100 mg/Kg SDG given orally (oral gavage or in drinking water) can induce Phase enzyme expression in lungs (see Fig. 41). Mice are sacrificed at 4, 6 and 9 months (see Fig.
- lung tissues are harvested for a) histological evaluation of tumor burden and quantification by image analysis, b) western blot detection of Nrf2- regulated AOE expression and oxidative stress, c) 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) levels in murine tissues and urine, and d) DNA adduct formation.
- a subset of mice are evaluated for lung inflammation using a) Bronchoalveolar lavage, and b) FACS analysis of inflammatory cells such as neutrophils, activated macrophages and T cells using antibodies to CD1 lb/Ly6G, CD1 lb/F4/80, and anti-CD3, respectively.
- SDG which should be detectable by measuring markers of oxidative stress.
- Nrf2 has two roles during carcinogenesis, one of which is preventive during tumor initiation and the second that promotes malignant progression.
- SDG is expected to be effective as a chemopreventive agent. Notwithstanding the role of Nrf2 in the anti- carcinogenic effects of SDG, it is possible that other mechanisms are also involved or even more important such as the modulation of miRNA in systemic fluids (blood) or lung tissues.
- the first group will consist of normal, healthy volunteers who have never smoked. This group allows assessment of true baseline levels (likely low) of genes of interest and oxidative biomarkers, in order to evaluate the changes induced by SDG supplementation in a "non-stimulated" environment.
- the second group of subjects will consist of current, active smokers. This group is selected because: i) they are at high risk for active cancer initiation and are potential subjects in a chemoprevention trial and ii) it has previously been shown that smoking induces its own genomic changes in the respiratory epithelium, as well as active oxidative stress.
- This group allows profiling of genomic and oxidative stress markers in smokers and to determine what genomic changes are induced by SDG in an already "activated” environment. This group also allows a determination of the reduction of elevated markers of oxidative stress by SDG in this high risk population.
- a crossover design is selected where each patient will also ingest both a placebo control and an SDG supplement both given in gelatin capsules (See Figure 43).
- Dosage A commercial preparation (BREVAILTM) was chosen to avoid the marked variability in SDG content and bioavailability observed with different batches of raw or ground flaxseed.
- synthetic SDG such as that described by Mishra et ah, Bioorganic & Medicinal Chemistry Letters 2013, (19):5325 will be used.
- Pharmacologic studies had shown that daily dosing with this formulation, which contains 50 mg of SDG, produced ENL levels (median, 63 nmol/L) similar to those found in the highest quintiles associated with reduction in cancer incidence in case-control studies.
- Clinical Study The study will be a single center, randomized, double-blind, two period cross-over trial. There will be two 3 week treatment periods interspersed with a one-month washout period ( Figure 43).
- the rationale for the cross-over design is that comparison of treatments within subjects removes any "subject effect" from the comparison and enhances the efficiency and power of the study.
- the study will be approved by the Penn Institutional Review Board, and all participants will provide written informed consent.
- a detailed questionnaire will be administered at baseline including a comprehensive smoking history, and environmental tobacco exposure (ETS). Participants will be seen weekly for evaluation of side-effects, adherence to test agent ingestion, collection of data on interim tobacco and medication use and sample collection.
- buccal mucosal epithelial cells are used as surrogate tissue to bronchial epithelial tissue. Such cells are easier to obtain (i.e. by using a simple swabbing of the cheek) than bronchial epithelial cells which require an invasive bronchoscopy.
- buccal epithelium can serve as surrogate tissue for airway bronchial tissues in gene array studies.
- HO-1 has also been implicated as a cytoprotective agent against oxidants and aromatic hydrocarbons in cigarette smoke in genetic studies. Therefore, HO-1 also represents a significant biomarker for monitoring the effects of flaxseed. Numerous studies have shown relationships between GST polymorphisms, smoking, and lung cancer. Importantly, benzo[a]pyrene-derived DNA-adducts in lung cells are regulated by detoxification of the reactive intermediate resulting from both cytochrome P-450- and aldo-keto-reductase- mediated metabolism by a lung-specific GST. As benzo[a]pyrene is a significant lung carcinogen and it is present in tobacco smoke, GSTs are monitored as biomarkers of the effects of flaxseed in smokers.
- BaP a carcinogen in cigarette smoke, causes reactive oxygen species-mediated DNA strand breaks and 8-oxo-7,8-dihydro-2'- deoxyguanosine (8-oxo-dGuo) formation.
- Exhaled Breath Condensate for evaluating lung oxidative stress: Given that it is not feasible to perform invasive tests like bronchoscopy in these subjects, a noninvasive sample collection technique, exhaled breath condensate (EBC) is used for investigating lung oxidant stress. Many substances are found in expired breath that are detectable in the liquid that can be obtained by cooling (i.e., condensing) it.
- EBC exhaled breath condensate
- the advantages of this method are that it is non-invasive and convenient, as a non-invasive sampling method for the real-time analysis and evaluation of oxidative stress biomarkers in lung.
- Biomarkers of oxidative stress include: H2O2, isoprostanes (IsoPs), malondialdehyde, 4-hydroxy-2-nonenal, antioxidants, glutathione and nitrosative stress markers.
- BaP (10 or 20 ⁇ ) induces ROS in a dose- and time-dependent manner when metabolically activated by epithelial cells (Figure 23).
- the immortalized CIO mouse bronchial cell line derived from Balb/C mice
- A549 cells a transformed, but highly differentiated lung cancer cell line used to model human type 1 alveolar epithelial cells- see data in Figures 22-23, and importantly, primary human bronchial epithelial cells.
- BaP-induced oxidative DNA damage caused by BaP can be measured by 8-oxo-dGuo formation, tail moment of individual cells using standard COMET image analysis (data not shown), or densitometric analysis of immunoblots for the detection of ⁇ 2 ⁇ (data not shown).
- a range of SDG concentrations is added at the same time as 10 ⁇ B[a]P and these parameters are measured.
- MTT cytotoxicity assays are performed to evaluate direct SDG effects on these cells in the absence and presence of the carcinogen (FS and FLC can only be tested in vivo).
- the ability of the purified SDG to upregulate Phase ⁇ enzymes in bronchial epithelial cells is tested.
- the first set of studies define the direct antioxidant effects of SDG.
- the second set of studies performed at the later time points when the SDG itself is gone (thus no direct antioxidant effects present), define the effects of SDG due to its Phase ⁇ enzyme-inducing effects.
- Additional measurements of oxidative stress and inflammation are performed to determine the decrease by SDG of plasma oxidative stress measurements (plasma malondialdehyde) and pro-inflammatory stress markers such as ( IL-6, IL-lot, ⁇ , TNF-ot, C-reactive protein).
- plasma oxidative stress measurements plasma oxidative stress measurements
- pro-inflammatory stress markers such as ( IL-6, IL-lot, ⁇ , TNF-ot, C-reactive protein).
- the animal studies generally contain more than two groups (e.g. control vs. 50mg/Kg SDG) of 20 mice and are analyzed using ANOVA or other suitable linear models. All calculations assume a two-sided test, an alpha level of 0.05, and at least 80% power.
- SDG or flaxseed diets are hypothesized to decrease asbestos induced ROS/inflammation leading to: 1) ROS, 2) decreased cytokines; 3) decreased HMGB1 ; 4) less tumorigenic foci; and 5) less tumors.
- the underlining hypothesis is that decreased inflammation and oxidative stress will lead to decreased malignant transformation of cells and less tumor burden (see Figure 48).
- ROS release Asbestos-Induced Oxidative Stress (ROS release) in Culture RAW Macrophages was evaluated. Cells were treated with the ROS-sensitive dye H2DCFDA for 30 min and then they were exposed to vehicle to 40 ⁇ g/cm2 asbestos fibers, or 4uM hypochlorite solution and fluorescence intensity was measured spectrophotometrically for 90, 150 minutes and 27 hours. Asbestos-induced ROS was generated shortly post asbestos exposure and continued for the duration of the observation period ( Figure 51).
- the in vitro experiments in this Example demonstrate that (1) SDG blocks asbestos-induced ROS in human mesothelial cells and mouse RAW macrophages; (2) SDG blocks inflammatory cytokine secretion by mouse peritoneal macrophages exposed to asbestos; and (3) SDG blocks oxidative (lipid peroxidation) and nitrosative stress (nitrite levels) in mouse peritoneal macrophages exposed to asbestos.
- SDG blocks asbestos-induced ROS in human mesothelial cells and mouse RAW macrophages
- SDG blocks inflammatory cytokine secretion by mouse peritoneal macrophages exposed to asbestos
- SDG blocks oxidative (lipid peroxidation) and nitrosative stress (nitrite levels) in mouse peritoneal macrophages exposed to asbestos.
- Flaxseed and its SDG-rich lignan component blunted asbestos-induced inflammation blunted asbestos-induced inflammation (younger mice) (Figure 60).
- Total white blood cells Figure 60A
- FS or FLC addition in the diet, albeit not significantly.
- levels were significantly blunted by both flaxseed and the SDG-lignan diet ( Figure 60B).
- Figure 62 Flaxseed lignan extract enriched in SDG (given in diet formulation) blunts asbestos inflammation in older mice ( Figure 62).
- Male NF2 (129SV) (+/-) mice were injected (intraperitoneal) with 400 ⁇ g of asbestos on Day 0. Mice were initiated on the test diets (0% FS or 10% FLC) the week prior to asbestos exposure (Day-7) and sacrificed on Day 3 post-asbestos exposure.
- Abdominal lavage (AL) was performed with 5 mL lx PBS (1 ml of belly lavage fluid was centrifuged and the supernatant was frozen). Plasma was collected at frozen at -80°. Cells were evaluated in lavage fluid and showed that total WBC and neutrophils, macrophages and eosinophils were all significantly decreased by the SDG-rich diet.
- Flaxseed lignan extract enriched in SDG blunts asbestos inflammatory cytokine secretion and nitrosative stress in older mice ( Figure 63).
- Male NF2 (129SV)(+/-) mice were injected (intraperitoneal) with 400 ⁇ g of asbestos on Day 0.
- Mice were initiated on test diets (0% FS or 10% FLC) the week prior to asbestos exposure (Day 7) and sacrificed on Day 3 post-asbestos exposure.
- Abdominal lavage (AL) was performed with 5 mL lx PBS (1 mL of belly lavage fluid was centrifuged and the supernatant frozen). Plasma was collected at frozen at -80°. Levels of cytokines ILi and TNFa as well as nitrites were significantly blunted by the SDG-rich diet.
- mice fed SDG-enriched diet had significantly reduced abdominal inflammation, as determined by abdominal lavage fluid WBCs; (2) SDG-enriched diet reduced the number of neutrophils in abdominal lavage fluid; (3) Levels of pro-inflammatory cytokines, IL- ⁇ and TNFa, were reduced in mice fed SDG-enriched; and (4) SDG-enriched diet- fed mice had lower levels of abdominal lavage fluid nitrite, indicative of reduced nitrosative stress induced by exposure to asbestos fibers.
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