JP2017520620A - Use of secoisolariciresinol diglucoside (SDG) and related compounds for protection against radiation and chemical damage - Google Patents

Abstract

The present invention relates to (S, S) -SDG, (R, R) -SDG, (S, R) -SDG, (R, S) -SDG, SDG, SECO, EL, ED, analogs thereof, these Compositions and methods for radioprotection and chemoprotection are provided using therapeutic and prophylactic methods using the stereoisomers and other related molecules. A method of protecting a biomolecule, cell, or tissue from radiation damage in a subject in need thereof, comprising administering to said subject an effective amount of at least one bioactive component, said bioactive component comprising: A method comprising secoisolarisiresinol diglucoside (SDG), secoisolarisiresinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or combinations thereof . [Selection] Figure 12

Description

Government Rights This invention is incorporated in part under grant numbers R01 (CA133470), 1P30ES013508-02, RC1AI081251 and 5-P30-CA-016520-34S2 awarded by the National Institutes of Health (NIH). It was made with support. The government has certain rights to the invention.

FIELD OF THE INVENTION Secoisolarisiresinol diglucoside (SDG), secoisolarisiresinol (SECO), enterodiol (ED), enterolactone (EL), their stereoisomers, their metabolites, and their similarities Compositions and methods are provided for radiation protection and radiation mitigation and chemoprotection using the body, such as from carcinogen-induced lung cancer and mesothelioma or from hypochlorite ions.

  Ionizing radiation has a wide range of harmful effects in living organisms. People are exposed to occupational accidents during diagnostic and therapeutic X-ray examination procedures, when using electronic equipment, from the background of nuclear accidents, during air and space travel, and long-term exposure to sunlight (eg, sunbathing). Exposed to radiation from a worker or outdoor worker). Exposure to natural radiation occurs in many forms. Natural resources such as air, water, and soil can be contaminated when in contact with natural radiation emitting materials (radionuclides), and radon is one such common source of natural radiation. In addition, current global development has established terrorism as a dangerous means of exposing many people to lethal radiation. Therefore, finding drugs that can be administered before or while exposed to radiation (ie, radioprotectors) and drugs as therapeutics after radiation exposure (ie, radiation mitigants) Is very important.

  In addition, lung cancer is a leading cause of cancer mortality in the United States. Despite new targeted therapies, improved tumor progression diagnostic techniques and surgical procedures, and increased use of combined chemoradiotherapy for locally advanced lung cancer, overall mortality declines Minimal (Tan & Spivack (2009) Lung Cancer 65: 129-137). Cancer chemoprevention is a dietary and pharmacological intervention that uses specific natural or synthetic drugs designed to prevent, inhibit, or reverse the carcinogenic process prior to the development of malignant lesions. Used (Hong & Sporn, (1997), Science 278: 1073-1077). One strategy for lung cancer chemoprotection focuses on the use of drugs that modulate the metabolism and pharmacokinetics of tobacco's environmental and other carcinogens through increased expression of detoxification phase 2 enzymes. Yes. Many synthetic and natural compounds are known to induce the expression of phase 2 enzymes. There have been many reports supporting the induction of Nrf2 / ARE-regulated phase 2 enzymes is a very effective strategy for reducing sensitivity to carcinogens. We have found that flaxseed (FS) and its main lignan SDG (both obtained by natural substance enrichment and synthesis) are effective lung cancer chemoprotective agents in a mouse model of chemically carcinogen-induced lung cancer. Data indicating that there is.

  About 85% of lung cancers are caused by smoking. The main lung carcinogens in tobacco smoke are polycyclic aromatic hydrocarbons represented by benzo [a] pyrene (BaP). Until better therapies are developed, the most promising ways to reduce mortality will be health checkups, smoking cessation, or prevention through chemoprotection. Chemoprotectants need to be administered for a long time to a large number of exposed but relatively healthy subjects. Chemoprotectants need to be safe, non-toxic, delicious and ideally affordable. Many chemoprotective agents have been studied in the field of lung cancer, but none meet these criteria. One of the most promising approaches is increased expression of phase 2 antioxidants and detoxification enzymes. Unfortunately, phase II enzyme activators such as oltipraz or sulforaphane that have been tested in patients to date have been found to be unacceptably toxic (Pendyala et al. (2001). Cancer Epidemiol Biomarkers). Prev 10: 269-272). Therefore, there is an urgent need for safe, non-toxic chemoprotective agents that are effective in preventing oxidative stress and DNA damage induced by lung carcinogens in tobacco smoke.

  Another well-known environmental carcinogen is asbestos, which refers to a group of natural hydrated fibrous silicate fibers that are used commercially for thermal insulation. In both animal models and patients, inhalation of asbestos fibers is associated with tumor diseases such as malignant mesothelioma (MM) and lung cancer (Carbon & Yang (2012). Clin Cancer Res 18: 598-604; Neri et al. 2012) Anticancer Res 32: 1005-1013), as well as clearly leading to pulmonary fibrosis. MM is a highly invasive cancer that arises from pleural and peritoneal mesothelial cells and has a median survival of about 1 year (Sterman et al. (2005). Clin Cancer Res 11, 7444-7453; Sterman et al. (1999) .Chest 116,504-520; Benard et al. (1999) .J Nucl Med 40,1241-1245). Other than surgery for very early disease, current therapies do not cure (Sterman & Albelda (2005). Respirology 10, 266-283). Currently, about 3,000 people die annually in the United States due to MM, and another 5,000 people die annually in Western Europe.

  Asbestos use is restricted in many Western countries, but it is still used in many countries around the world, and it is estimated that more than 2 million tons were mined in 2008 (Survey, B (G. (2010). World Mineral Production 2004-08. Nottingham, UK, British Geologic Survey). Thus, there can be a dramatic increase in the number of cases of MM in the third world (especially India) where asbestos use is increasing with few precautions. However, significant exposure still exists in developed countries. These include working on existing asbestos (ie lead pipes, piping, insulators, insulator removal, etc.) and super funds (funds for removal of neglected hazardous industrial waste) asbestos hazardous waste treatment plants, etc. Includes many types of occupations that expose people. There are also environmental and domestic exposures. For example, there is an increased risk of MM in areas where mining or asbestos plants are closed.

  An important issue in the association between asbestos and cancer is that inhaled asbestos fibers remain in the lungs for an extremely long time, thereby causing continuous damage even if the patient leaves the exposure . Because of this long incubation period (often 30-50 years), previously exposed individuals remain at high risk for MM and other cancers throughout their lives.

Cancer chemoprotection aims to prevent, stop, or reverse either the onset of carcinogenesis or the progression of neoplastic cells to cancer. While this definition sounds simple, finding an effective chemoprotectant has been very difficult. First, the mechanisms by which carcinogens induce cancer usually involve multiple mechanisms, making it difficult to achieve efficacy and requiring agents with multiple activities. Second, it is extremely non-toxic, well tolerated, and affordable because it is used to prevent a small number of tumors in a large population of healthy but at risk individuals There is a need.
Therefore, drugs that can be administered before or during exposure to other harmful chemicals such as carcinogens or chemical warfare agents, chlorine and hypochlorite ions and other harmful poisons (i.e. chemical Finding a defensive agent is also extremely important.

Tan & Spivak (2009) Lung Cancer 65: 129-137 Hong & Sporn, (1997), Science 278: 1073-1077. Pendyala et al. (2001). Cancer Epidemiol Biomarkers Prev 10: 269-272 Carbone & Yang (2012). Clin Cancer Res 18: 598-604 Neri et al. (2012). Anticancer Res 32: 1005-1013 Sterman et al. (2005). Clin Cancer Res 11, 7444-7453 Sterman et al. (1999). Chest 116,504-520 Benard et al. (1999). J Nucl Med 40, 1241-1245 Sterman & Albelda (2005). Respirology 10,266-283 Surveyy, B.M. G. (2010). World Mineral Production 2004-08. Nottingham, UK, British Geologic Survey

  In one aspect, the invention relates to a method of protecting a biomolecule (such as genetic material such as a nucleic acid, protein or lipid), cell, or tissue from radiation damage in a subject in need thereof, Administering to said subject an effective amount of flaxseed, its bioactive ingredients, its degradation products or metabolites. Administration to the subject includes administration before, during and after exposure to harmful radiation exposure. The time before, during and after exposure can be seconds, minutes, hours, days, weeks, months or even years. The bioactive component includes secoisolaricillinol diglucoside (SDG), secoisolaricillinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or These combinations are included.

  In another aspect, the invention relates to a method of treating or preventing radiation damage in a subject that has been or will be exposed to radiation, said method comprising an effective amount of at least one of said subject Administration of a physiologically active ingredient, wherein the physiologically active ingredient is secoisolarisiresinol diglucoside (SDG), secoisolarisiresinol (SECO), enterodiol (ED), enterolactone (EL), and the like Body, these stereoisomers, or a combination thereof.

  In another aspect, the invention protects biomolecules (such as genetic material such as nucleic acids, proteins or lipids), cells, or tissues in a subject in need thereof from radiation damage resulting from accidental radiation exposure. With respect to the method, the method comprises administering to the subject an effective amount of flaxseed, its bioactive component, its degradation product or metabolite.

  In another aspect, the invention relates to a method of protecting biomolecules (such as genetic material such as nucleic acids, proteins or lipids), cells, or tissues from radiation damage that causes aging.

  In another aspect, the invention relates to radiation damage resulting from exposure of biomolecules (such as genetic material such as nucleic acids, proteins or lipids), cells, or tissues to chemical carcinogens and toxins derived from both natural and synthetic sources. On how to protect against.

  In another aspect, the invention relates to radiation damage resulting from radiation therapy for cancer treatment of biomolecules (such as genetic material such as nucleic acids, proteins or lipids), cells, or tissues in a subject in need thereof. The method includes administering to the subject an effective amount of flaxseed, its bioactive component, its degradation product or metabolite.

  In another aspect, the invention relates to a method of protecting a biomolecule (such as genetic material such as a nucleic acid, protein or lipid), cell or tissue in a subject in need thereof from radiation damage. Administering an effective amount of at least one bioactive ingredient to the subject, wherein the bioactive ingredient comprises secoisolarisiresinol diglucoside (SDG), secoisolarisiresinol (SECO), enterodiol (ED). ), Enterolactone (EL), analogs thereof, stereoisomers thereof, or combinations thereof. In some embodiments, the radiation damage results from accidental radiation exposure. In some embodiments, the radiation damage results from radiation therapy for cancer (eg, lung cancer) treatment.

  In another aspect, the present invention relates to a method for preventing radiation-induced damage to a biomolecule (such as genetic material such as nucleic acids, proteins or lipids), cells, or tissues in a subject in need thereof. Includes administering to the subject an effective amount of flaxseed, a physiologically active ingredient thereof, or a metabolite thereof.

  In another aspect, the present invention relates to a method for preventing radiation-induced damage to a biomolecule (such as genetic material such as nucleic acids, proteins or lipids), cells, or tissues in a subject in need thereof. Comprises administering to the subject an effective amount of at least one physiologically active ingredient, wherein the physiologically active ingredient is secoisolarisiresinol diglucoside (SDG), secoisolarisiresinol (SECO), enterodiol ( ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or combinations thereof.

  In another aspect, the invention relates to a method of protecting a biomolecule (such as genetic material such as a nucleic acid, protein or lipid), cell or tissue from radiation damage to the cell, said method comprising an effective amount of at least one Contacting said cell with said bioactive component, said bioactive component comprising secoisolaricillinol diglucoside (SDG), secoisolaricillinol (SECO), enterodiol (ED), enterolactone (EL) ), Analogs thereof, stereoisomers thereof, or combinations thereof.

  In another aspect, the invention relates to a method of protecting a biomolecule (such as genetic material such as a nucleic acid, protein or lipid), cell, or tissue in a subject in need thereof from damage by a carcinogen, This method comprises administering to the subject an effective amount of flaxseed, a bioactive component thereof, a degradation product or a metabolite thereof. Administration to the subject includes administration before, during and after adverse exposure to both natural and synthetic chemical carcinogens and toxicants. The time before, during and after exposure can be seconds, minutes, hours, days, weeks, months or even years. The bioactive component includes secoisolaricillinol diglucoside (SDG), secoisolaricillinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or These combinations are included.

  In another aspect, the invention relates to a method of protecting biomolecules in a subject in need thereof from damage by carcinogens resulting from accidental exposure to chemical and carcinogens and toxins from both natural and synthetic sources, This method comprises administering to the subject an effective amount of flaxseed, a bioactive component thereof, a degradation product or a metabolite thereof.

  In another aspect, the present invention relates to chemical carcinogens derived from both natural and synthetic biomolecules (such as genetic material such as nucleic acids, proteins or lipids), cells, or tissues that cause lung cancer or mesothelioma. And a method for protecting against radiation damage from poisons.

  In another aspect, the invention relates to a method of protecting a biomolecule (such as genetic material such as a nucleic acid, protein or lipid), cell, or tissue from damage by a carcinogen, the method comprising: Contacting the cell or tissue with an effective amount of a physiologically active ingredient. Contact with the biomolecule, cell, or tissue includes contact before, during, and after harmful exposure to both natural and synthetic chemical carcinogens and toxicants. The time before, during and after exposure can be seconds, minutes, hours, days, weeks, months or even years. The bioactive component includes secoisolaricillinol diglucoside (SDG), secoisolaricillinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or These combinations are included.

  In another aspect, the invention provides a carcinogen-induced injury caused by a carcinogen-induced cancer in a subject that has been or will be exposed to one or more carcinogens. Relates to a method for treating or preventing malignant transformation or cancer development, the method comprising administering to the subject an effective amount of at least one bioactive component, wherein the bioactive component is secoisolarisiresinol diglucoside. (SDG), secoisolaricillinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or combinations thereof.

  In another aspect, the invention relates to a method of protecting a subject exposed to one or more carcinogens from a carcinogen-induced cancer, wherein the method comprises an effective amount of flaxseed, its physiological Administration of the active ingredient, its metabolites.

  In another aspect, the present invention provides a method of protecting a biomolecule (such as genetic material such as a nucleic acid, protein or lipid), cell, or tissue in a subject in need thereof from damage by hypochlorite ions. In this regard, this method comprises administering to the subject an effective amount of flaxseed, a bioactive component thereof, a degradation product or a metabolite thereof. Administration to the subject includes administration before, during and after adverse exposure to both natural and synthetic chemical carcinogens and toxicants. The time before, during and after exposure can be seconds, minutes, hours, days, weeks, months or even years. The bioactive component includes secoisolaricillinol diglucoside (SDG), secoisolaricillinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or These combinations are included.

  In another aspect, the invention relates to a method of treating or preventing hypochlorite ion-induced damage in a subject that has been or will be exposed to hypochlorite ion. Comprises administering to the subject an effective amount of at least one physiologically active ingredient, wherein the physiologically active ingredient is secoisolarisiresinol diglucoside (SDG), secoisolarisiresinol (SECO), enterodiol ( ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or combinations thereof.

  In another aspect, the invention relates to a method of protecting biomolecules (such as genetic material such as nucleic acids, proteins or lipids), cells, or tissues from damage by hypochlorite ions, the method comprising: Contacting the biomolecule, cell, or tissue exposed to, or expected to be exposed to, chlorate ions with an effective amount of a bioactive component. Contact with the biomolecule, cell, or tissue includes contact before, during, and after harmful exposure to both natural and synthetic chemical carcinogens and toxicants. The time before, during and after exposure can be seconds, minutes, hours, days, weeks, months or even years. The bioactive component includes secoisolaricillinol diglucoside (SDG), secoisolaricillinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or These combinations are included.

  In another aspect, the invention relates to a composition for use in one of the aforementioned methods.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Other features and advantages of the present invention will become apparent from the following detailed description and drawings. However, the detailed description and specific examples, while indicating the preferred embodiment of the invention, are provided by way of illustration only and are within the spirit and scope of the invention. It should be understood that various changes and modifications will be apparent to those skilled in the art. Also, whenever embodiments are described under different aspects of the present invention, whenever appropriate, any embodiment of the present invention may include one or more other implementations of the present invention. It is also contemplated that it can be combined with a form.

A-B. Effect of increasing gamma dose on plasmid (pBR322) DNA relaxation. The super coil (SC) represents a compact type. The open coil (OC) type represents a relaxed or damaged plasmid. The (A) -supercoil (SC) type is the lower clear band (at the position of 3,000 bp), and the open coil (OC) type is the upper clear band. Lane 1-1 kb DNA standard ladder, lanes 2 and 3 untreated plasmid DNA, plasmid DNA exposed to lanes 4 and 5-10 Gy, plasmid DNA exposed to lanes 6 and 7-25 Gy, lanes 8 and 9-50 Gy exposed Plasmid DNA. (B) -SC and OC types are expressed as a percentage of total plasmid DNA. All samples were tested twice at each condition. Data are expressed as mean ± standard deviation. p <0.05 was considered significant. * And ** indicate that there is a significant difference when compared to untreated SC and OC types, respectively. A-I. Effect of increasing concentrations of synthetic SDG (S, S), SDG (R, R) and commercial SDG on gamma-induced plasmid (pBR322) DNA relaxation. All samples were exposed to a gamma dose of 25 Gy. SDG concentrations were 25, 50, 100 and 250 μM. Figures (A), (D), and (G)-plasmids after exposure to 25 Gy in the presence of 25, 50, 100 and 250 μM SDG (S, S), SDG (R, R) and SDG (commercially available) A representative agarose gel scan of DNA is shown. Lane 1-1 kb DNA standard ladder, lanes 2 and 3 untreated plasmid DNA, lanes 4 and 5-25 μM, lanes 6 and 7-50 μM, lanes 8 and 9-100 μM, and lanes 10 and 11-250 μM SDG. Figures (B), (E) and (H) -SC and OC types are expressed as a percentage of total plasmid DNA. All samples were tested twice at each condition. Data were expressed as mean ± standard deviation. p <0.05 was considered significant. * And # indicate that there is a significant difference when compared to untreated SC and OC types, respectively. ** and ## indicate that there is a significant difference when compared to samples exposed to 25 Gy without SDG treatment. Figures (C), (F) and (I) show SDG-dependent suppression of plasmid DNA relaxation. EC ₅₀ values were determined from the quadratic equation shown below the curve. A-B. Effect of increasing gamma dose on calf thymus DNA fragmentation. Small molecular weight fragments are produced by DNA exposure to gamma radiation. Small molecular weight fragments move faster than higher molecular weight DNA. Measurement of the density of low molecular weight DNA fragments (<6,000 bp) compared to high molecular weight DNA (> 6,000 bp) reflects the extent of radiation-induced damage. (A) Lane 1-1 kb DNA standard ladder, lanes 2 and 3 untreated calf thymus DNA, DNA exposed to lanes 4 and 5-25 Gy, DNA exposed to lanes 6 and 7-50 Gy. (B)-High and low molecular weight DNA types are expressed as a percentage of total DNA. All samples were tested twice at each condition. Data were expressed as mean ± standard deviation. p <0.05 was considered significant. * Indicates that there is a significant difference when compared to no treatment, respectively. A-F. Effect of increasing concentrations of synthetic SDG (S, S), SDG (R, R) and commercial SDG on gamma-induced calf thymus DNA fragmentation. All samples were exposed to a 50 gray gamma dose. SDG concentrations were 25, 50, 100 and 250 μM. Figures (A), (C), and (E) -25 pups after exposure to 50 Gy in the presence of 25, 50, 100 and 250 μM SDG (S, S), SDG (R, R) and SDG (commercially available) A representative agarose gel scan of bovine thymus DNA is shown. Lane 1-1 kb DNA standard ladder, lanes 2 and 3 untreated DNA, lanes 4 and 5-25 μM, lanes 6 and 7-50 μM, lanes 8 and 9-100 μM, and lanes 10 and 11-250 μM SDG. Figures (B), (D) and (F)-high and low molecular weight DNA types are expressed as percent of total DNA. All samples were tested twice at each condition. Data were expressed as mean ± standard deviation. p <0.05 was considered significant. * Indicates that there is a significant difference when compared to untreated DNA. # Indicates that there is a significant difference when compared to the sample exposed to 50 Gy without SDG treatment. A-F. Effect of very low concentrations of synthetic SDG (S, S), SDG (R, R) and commercial SDG on gamma-induced calf thymus DNA fragmentation. All samples were exposed to a 50 Gy gamma dose. SDG concentrations were 0.5, 1.0, 5.0 and 10 μM. Figures (A), (C), and (E)-in the presence of 0.5, 1.0, 5.0 and 10 μM SDG (S, S), SDG (R, R) and SDG (commercially available) A representative agarose gel scan of calf thymus DNA after exposure to 50 Gy is shown. Lane 1-1 kb DNA standard ladder, lanes 2 and 3-untreated DNA, lanes 4 and 5-0.5 μM, lanes 6 and 7-1.0 μM, lanes 8 and 9-5.0 μM, and lanes 10 and 11- 10 μM SDG. Figures (B), (D) and (F)-high and low molecular weight DNA types are expressed as percent of total DNA. All samples were tested twice at each condition. Data were expressed as mean ± standard deviation. p <0.05 was considered significant. * Indicates that there is a significant difference when compared to untreated DNA. # Indicates that there is a significant difference when compared to the sample exposed to 50 Gy without SDG treatment. A-B. Effect of SDG, SECO, ED and EL on gamma-induced calf thymus DNA fragmentation. All samples were exposed to a 50 Gy gamma dose. SDG, SECO, ED and EL were used at a concentration of 10 μM. (A) Representative agarose gel scan of calf thymus DNA after exposure to 50 Gy in the presence of 10 μM SDG, SECO, ED and EL. Lane 1-1 kb DNA standard ladder, lanes 2 and 3 untreated DNA, lanes 4, 5 and 6-IR 50 Gy, lanes 7 and 8-SDG, lanes 9 and 10-SECO, lanes 11 and 12-ED, and lane 13 and 14-EL. (B) High and low molecular weight DNA types are expressed as a percentage of total DNA. All samples were tested twice at each condition. Data were expressed as mean ± standard deviation. p <0.05 was considered significant. * Indicates that there is a significant difference when compared to untreated DNA. # Indicates that there is a significant difference when compared to samples exposed to 50 Gy alone. A-C. Effect of SDG treatment on radiation dose response of mouse primary lung cells. (A) epithelial cells; (B) endothelial cells and (C) WT fibroblasts. Prior to gamma irradiation (0, 2, 4, 6, 8 Gy), cells were treated with different concentrations of SDG for 6 hours and incubated. All visible colonies were counted on days 12-14 and viability was normalized to control values. Data were expressed as mean ± standard error. For irradiated cells versus 50 μM SDG pretreated irradiated cells, ** is p ≦ 0.001, * is p ≦ 0.01, # is p ≦ 0.05. A-B. Assessment of radiation-induced DNA single strand breaks (SSB) in lung cells using an alkaline comet assay. (A) Kinetic assessment of DNA damage in primary lung cells (epithelium, endothelium and WT fibroblasts) irradiated with 2 Gy; at least 100-150 cells were counted for each treatment. DNA damage was assessed by calculating the “tail moment” (the product of the amount of DNA in the tail and the tail length) for each cell. For unirradiated controls vs. their respective irradiated cells, * is p ≦ 0.001; (B) SDG (50 μM) treatment of irradiated primary lung cells (treatment at 0, 2, 4, 6 hours prior to irradiation). effect. Data were expressed as mean ± standard error. For irradiated cells versus SDG pretreated irradiated cells, * is p ≦ 0.001, # is p ≦ 0.01. Inset: Representative fluorescence micrograph of primary lung epithelial cells. Cells were pretreated with SDG (50 μM), exposed to gamma rays (2 Gy), embedded in agarose, lysed, electrophoresed (0.66 V / cm, 25 minutes), stained with SYBR green, and fluorescent After visualization under the microscope, comet tail formation was examined. (A) Control cells, (B) SDG (50 μM), (C) Irradiated cells after 30 minutes exposure to IR (2 Gy), (D) Pretreated with SDG (50 μM, 6 hours). A-C. Fluorescence assessment of γ-H2AX focus induction in irradiated mouse primary lung cells, ie epithelial cells, endothelial cells and WT fibroblasts. The cells were treated with SDG (50 μM) for 6 hours and irradiated with γ rays (2 Gy). At desired time intervals, cells were fixed with 4% paraformaldehyde, washed, detected with γ-H2AX antibody, and cell nuclei were counterstained with DAPI. Cells were visualized under a fluorescence microscope. Total cells (blue), γ-H2AX positive cells (green) were counted per field and the percentage of γ-H2AX positive cells was calculated. At least 500 cells were counted for each treatment and experiments were performed twice. Data were expressed as mean ± standard error. For irradiated cells versus SDG pretreated irradiated cells, * is p ≦ 0.05, ** is p ≦ 0.005. Representative panel of immunofluorescence visualization of γ-H2AX focus (green) in mouse lung epithelial cells. Cells were pretreated with SDG for 6 hours, then γ-irradiated (2 Gy) and incubated for an additional 30 minutes. Cells were fixed with 4% paraformaldehyde and detected with γ-H2AX antibody. DNA was counterstained with DAPI (blue). Images were acquired using a fluorescence microscope. A-C. Flow cytometry (FACS) confirmation of γ-H2AX focus induction in irradiated mouse primary lung cells. Primary lung cells, ie, epithelial cells (A), endothelial cells (B) and WT fibroblasts (C) were treated with SDG (50 μM) for 6 hours and γ-irradiated (2 Gy). Cells were processed for FACS analysis at desired time intervals. Data were quantified using Summit software and expressed as mean ± standard error. For irradiated cells versus SDG pretreated irradiated cells, * is p ≦ 0.05, ** is p ≦ 0.01. A-C. Assessment of radiation-induced apoptotic death in primary lung cells. Quantitative evaluation of the effect of SDG (50 mM) pretreatment (6 hours) on irradiated primary lung cells, ie epithelial cells (A), endothelial cells (B) and WT fibroblasts (C). Cells were fixed, stained with DAPI and visualized for morphological analysis under a fluorescence microscope. For each treatment, at least 500 cells from 5 different fields were counted and the percentage of apoptotic cells was calculated. The experiment was performed twice. Data were expressed as mean ± standard error. For irradiated cells versus SDG pretreated irradiated cells, * is p ≦ 0.05, ** is p ≦ 0.005. Evaluation of the effect of SDG treatment on the regulators of apoptosis in AE lung epithelial cells. Mouse primary lung cells (epithelial cells) were treated with SDG (50 μM) for 6 hours and then exposed to 2 Gy. Cells were harvested at 6, 24, and 48 hours after incubation. Total RNA was isolated from epithelial cells at desired time intervals and Bax and Bcl-2 gene expression was assessed by quantitative real-time RT-PCR analysis (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 image (C) and densitometric analysis normalized to β-actin (D and E). Data were expressed as mean ± standard error. For IR exposed cells compared to SDG treated cells or SDG + IR treated cells, * is p ≦ 0.05, ** is p ≦ 0.01. A-C. Effect of SDG on radiation-induced increase in levels of active caspase 3 and cleaved PARP. Mouse primary lung cells (epithelial cells) were treated with SDG (50 μM) for 6 hours and then exposed to 2 Gy. Cells were harvested at 6, 24, and 48 hours after irradiation. Cleaved caspase 3 and cleaved PARP protein levels were assessed by Western blot analysis. (A) Representative images and (B and C) densitometric analysis normalized to β-actin. The analysis was repeated twice and the data were expressed as mean ± standard error. For IR exposed cells compared to SDG treated cells, * is p ≦ 0.05, ** is p ≦ 0.01. A-B. SDG removes hypochlorite ions. FIG. 15A shows a ClO ^- dependent increase in APF and HPF fluorescence. FIG. 15B shows the removal effect of ClO ⁻ by SDG. FIG. 15C shows the removal effect of synthetic SDG diastereomers SDG (S, S) and SDG (R, R). All samples were tested twice. Data were expressed as mean ± standard deviation. p <0.05 was considered significant. * Indicates a significant difference when compared to the untreated control. SDG removes hypochlorite ions. FIG. 15A shows a ClO ^- dependent increase in APF and HPF fluorescence. FIG. 15B shows the removal effect of ClO ⁻ by SDG. FIG. 15C shows the removal effect of synthetic SDG diastereomers SDG (S, S) and SDG (R, R). All samples were tested twice. Data were expressed as mean ± standard deviation. p <0.05 was considered significant. * Indicates a significant difference when compared to the untreated control. A-B. SDG eliminates hypochlorite production induced by gamma radiation. FIGS. 16A and B show the increase in APF and HPF fluorescence by γ-ray induction. FIGS. 16C and D show the effect of SDG on hypochlorite production in phosphate buffered saline (PBS) containing APF or HPF at increasing radiation doses (see legend in FIG. 15). Figures 16E, F and G show gamma-induced chlorination of taurine. FIG. 16E shows the hypochlorite dependent chlorination of taurine. FIG. 16F shows the absorbance of taurine chloramine for all experimental conditions. FIG. 16G shows hypochlorite concentration under various conditions similar to FIG. 16F. For FIGS. 16A-E, all samples were run twice and for FIGS. 16F and G, all samples were run 4 times. Data were expressed as mean ± standard deviation. p <0.05 was considered significant. * Indicates a significant difference when compared to the untreated control. CD. SDG eliminates hypochlorite production induced by gamma radiation. FIGS. 16A and B show the increase in APF and HPF fluorescence by γ-ray induction. FIGS. 16C and D show the effect of SDG on hypochlorite production in phosphate buffered saline (PBS) containing APF or HPF at increasing radiation doses (see legend in FIG. 15). Figures 16E, F and G show gamma-induced chlorination of taurine. FIG. 16E shows the hypochlorite dependent chlorination of taurine. FIG. 16F shows the absorbance of taurine chloramine for all experimental conditions. FIG. 16G shows hypochlorite concentration under various conditions similar to FIG. 16F. For FIGS. 16A-E, all samples were run twice and for FIGS. 16F and G, all samples were run 4 times. Data were expressed as mean ± standard deviation. p <0.05 was considered significant. * Indicates a significant difference when compared to the untreated control. A-B. SDG eliminates hypochlorite production induced by gamma radiation. FIGS. 16A and B show the increase in APF and HPF fluorescence by γ-ray induction. FIGS. 16C and D show the effect of SDG on hypochlorite production in phosphate buffered saline (PBS) containing APF or HPF at increasing radiation doses (see legend in FIG. 15). Figures 16E, F and G show gamma-induced chlorination of taurine. FIG. 16E shows the hypochlorite dependent chlorination of taurine. FIG. 16F shows the absorbance of taurine chloramine for all experimental conditions. FIG. 16G shows hypochlorite concentration under various conditions similar to FIG. 16F. For FIGS. 16A-E, all samples were run twice and for FIGS. 16F and G, all samples were run 4 times. Data were expressed as mean ± standard deviation. p <0.05 was considered significant. * Indicates a significant difference when compared to the untreated control. FG. SDG eliminates hypochlorite production induced by gamma radiation. FIGS. 16A and B show the increase in APF and HPF fluorescence by γ-ray induction. FIGS. 16C and D show the effect of SDG on hypochlorite production in phosphate buffered saline (PBS) containing APF or HPF at increasing radiation doses (see legend in FIG. 15). Figures 16E, F and G show gamma-induced chlorination of taurine. FIG. 16E shows the hypochlorite dependent chlorination of taurine. FIG. 16F shows the absorbance of taurine chloramine for all experimental conditions. FIG. 16G shows hypochlorite concentration under various conditions similar to FIG. 16F. For FIGS. 16A-E, all samples were run twice and for FIGS. 16F and G, all samples were run 4 times. Data were expressed as mean ± standard deviation. p <0.05 was considered significant. * Indicates a significant difference when compared to the untreated control. A-B. Hypochlorite-induced calf thymus DNA damage. FIGS. 17A and C show representative agarose gel scans of calf thymus DNA after exposure to HOCl. Figures 17B and D show high and low molecular weight DNA fragments as a percentage of total DNA. Figures 17E and F show the effect of SDG on hypochlorite-induced damage to plasmid DNA. FIG. 17E shows a representative agarose gel of plasmid DNA after exposure to HOCl. FIG. 17F shows SC and OC types expressed as a percentage of total plasmid DNA. In FIG. 17A, the lane 1-1 kb DNA standard ladder, lanes 2 and 3-untreated DNA, lanes 4 and 5-0.1 mM, lanes 6 and 7-0.2 mM, lanes 8 and 9-0.4 mM, lane 10 and 11-0.5MM, and lanes 12 and 13-0.6mM ClO ^- it is. In FIG. 17C, lane 1-1 kb DNA standard ladder, lanes 2 and 3 untreated DNA, lanes 4 and 5-0.5 mM HOCl, lanes 6 and 7-0.5 mM HOCl + SDG (commercially available) 1 μM, lanes 8 and 9− 0.5 mM HOCl + SDG (S, S) 1 μM, lanes 10 and 11-0.5 mM HOCl + SDG (R, R) 1 μM, lanes 12 and 13-0.5 mM HOCl + quercetin 1 μM, and lanes 14 and 15-0.5 mM HOCl + silibinin 1 μM. In FIG. 17E, lane 1-1 kb DNA standard ladder, lanes 2 and 3-untreated DNA, lanes 4 and 5-4.5 mM HOCl, lanes 6 and 7-4.5 mM HOCl + SDG 25 μM. All samples were tested twice at each condition. Data were expressed as mean ± standard deviation. p <0.05 was considered significant. * And # indicate that there is a significant difference when compared to the untreated control. CD. Hypochlorite-induced calf thymus DNA damage. FIGS. 17A and C show representative agarose gel scans of calf thymus DNA after exposure to HOCl. Figures 17B and D show high and low molecular weight DNA fragments as a percentage of total DNA. Figures 17E and F show the effect of SDG on hypochlorite-induced damage to plasmid DNA. FIG. 17E shows a representative agarose gel of plasmid DNA after exposure to HOCl. FIG. 17F shows SC and OC types expressed as a percentage of total plasmid DNA. In FIG. 17A, the lane 1-1 kb DNA standard ladder, lanes 2 and 3-untreated DNA, lanes 4 and 5-0.1 mM, lanes 6 and 7-0.2 mM, lanes 8 and 9-0.4 mM, lane 10 and 11-0.5MM, and lanes 12 and 13-0.6mM ClO ^- it is. In FIG. 17C, lane 1-1 kb DNA standard ladder, lanes 2 and 3 untreated DNA, lanes 4 and 5-0.5 mM HOCl, lanes 6 and 7-0.5 mM HOCl + SDG (commercially available) 1 μM, lanes 8 and 9− 0.5 mM HOCl + SDG (S, S) 1 μM, lanes 10 and 11-0.5 mM HOCl + SDG (R, R) 1 μM, lanes 12 and 13-0.5 mM HOCl + quercetin 1 μM, and lanes 14 and 15-0.5 mM HOCl + silibinin 1 μM. In FIG. 17E, lane 1-1 kb DNA standard ladder, lanes 2 and 3-untreated DNA, lanes 4 and 5-4.5 mM HOCl, lanes 6 and 7-4.5 mM HOCl + SDG 25 μM. All samples were tested twice at each condition. Data were expressed as mean ± standard deviation. p <0.05 was considered significant. * And # indicate that there is a significant difference when compared to the untreated control. EF. Hypochlorite-induced calf thymus DNA damage. FIGS. 17A and C show representative agarose gel scans of calf thymus DNA after exposure to HOCl. Figures 17B and D show high and low molecular weight DNA fragments as a percentage of total DNA. Figures 17E and F show the effect of SDG on hypochlorite-induced damage to plasmid DNA. FIG. 17E shows a representative agarose gel of plasmid DNA after exposure to HOCl. FIG. 17F shows SC and OC types expressed as a percentage of total plasmid DNA. In FIG. 17A, the lane 1-1 kb DNA standard ladder, lanes 2 and 3-untreated DNA, lanes 4 and 5-0.1 mM, lanes 6 and 7-0.2 mM, lanes 8 and 9-0.4 mM, lane 10 and 11-0.5MM, and lanes 12 and 13-0.6mM ClO ^- it is. In FIG. 17C, lane 1-1 kb DNA standard ladder, lanes 2 and 3 untreated DNA, lanes 4 and 5-0.5 mM HOCl, lanes 6 and 7-0.5 mM HOCl + SDG (commercially available) 1 μM, lanes 8 and 9− 0.5 mM HOCl + SDG (S, S) 1 μM, lanes 10 and 11-0.5 mM HOCl + SDG (R, R) 1 μM, lanes 12 and 13-0.5 mM HOCl + quercetin 1 μM, and lanes 14 and 15-0.5 mM HOCl + silibinin 1 μM. In FIG. 17E, lane 1-1 kb DNA standard ladder, lanes 2 and 3-untreated DNA, lanes 4 and 5-4.5 mM HOCl, lanes 6 and 7-4.5 mM HOCl + SDG 25 μM. All samples were tested twice at each condition. Data were expressed as mean ± standard deviation. p <0.05 was considered significant. * And # indicate that there is a significant difference when compared to the untreated control. A-B. Effect of SDG (before and after treatment) on hypochlorite-induced modification of 2-aminopurine (2-AP). FIG. 18A shows a representative spectrum for all conditions. FIG. 18B shows the fluorescence at 374 nm under different conditions similar to FIG. 18A. FIG. 18C shows% protection by SDG. All samples were tested twice at each condition. Data were expressed as mean ± standard deviation. p <0.05 was considered significant. * And # indicate that there is a significant difference when compared to untreated 2-AP control and treatment, respectively. Effect of SDG (before and after treatment) on hypochlorite-induced modification of 2-aminopurine (2-AP). FIG. 18A shows a representative spectrum for all conditions. FIG. 18B shows the fluorescence at 374 nm under different conditions similar to FIG. 18A. FIG. 18C shows% protection by SDG. All samples were tested twice at each condition. Data were expressed as mean ± standard deviation. p <0.05 was considered significant. * And # indicate that there is a significant difference when compared to untreated 2-AP control and treatment, respectively. A-B. SDG prevents γ-ray-induced modification of 2-aminopurine (2-AP). FIG. 19A shows a representative spectrum for all conditions. FIG. 19B shows the fluorescence at 374 nm. All samples were tested twice at each condition. Data were expressed as mean ± standard deviation. p <0.05 was considered significant. * And # indicate that there is a significant difference when compared to untreated 2-AP control and treatment, respectively. Proposed mechanism for the action of SDG in protecting DNA from nucleobase chlorination. Mechanism of chemical defense by flaxseed and its lignans. SDG reduces lung tumor formation due to tobacco and other environmental carcinogens by inhibiting a multi-stage carcinogenic process. We provide evidence that lignan SDG has chemoprotective activity in both animal models, through modulation of the Nrf2-regulated phase 2 detoxification pathway, and possibly other mechanisms. We further provide data to support that the protective effect of SDG is mediated by direct ROS ablation and / or indirect antioxidant / anti-inflammatory properties, as well as reduced carcinogen toxicity and DNA damage. SDG reduces DNA oxidative damage induced by benzoalphapyrene in cells. SDG (10 μM) is added to human epithelial cells (A549) exposed to 25 μM tobacco and the environmental carcinogen benzoalphapyrene (BaP) and mass spectrometry is used to determine 8-oxo-7,8-dihydroguanine ( Oxidative damage to DNA was detected as indicated by the presence of 8-oxo-dGuo). SDG reduced DNA damage at 3 and 6 hours after carcinogen exposure. Figure 2 shows the carcinogen benzoalphapyrene-induced ROS in cells. As detected by the redox sensitive fluorescent dye, exposure of mouse epithelial cells to BaP induces harmful reactive oxygen species (ROS). As early as 2 hours after exposure to carcinogens, a clear increase in fluorescence intensity indicates the production of ROS in the cells. SDG prevents ROS generation by carcinogen exposure. Mouse epithelial cells were exposed to 10 or 20 μM BaP and increasing concentrations of SDG (0, 0.1, 0.5, 1, 5 μM SDG), and 2 hours later (time judged reasonable from FIG. 23). ROS was detected. SDG removed harmful ROS to a negligible level. SDG prevents genotoxic stress in human epithelial cells exposed to BaP. Exposure of cells to potent carcinogens such as BaP induces genotoxic stress indicated by increased levels of p53 protein. This is alleviated in a dose-dependent manner by the presence of SDG at concentrations of 5, 10, 25 and 50 μM. SDG prevents oxidative DNA damage in human epithelial cells exposed to BaP. Exposure of cells to potent carcinogens such as BaP induces oxidative DNA damage as indicated by increased levels of γ-H2AX, a marker for double-stranded DNA breaks. This is mitigated in a dose-dependent manner by the presence of SDG at concentrations of 5, 10, 25, 50 and 100 μM. SDG prevents DNA adduct formation in human epithelial cells exposed to BaP. Exposure of cells to strong carcinogens such as BaP induces the formation of DNA adducts. A DNA adduct is a fragment of DNA covalently bound to a carcinogen and is directly associated with the development of malignant lesions. DNA adduct levels are reduced by the presence of SDG or its metabolites ED and EL added alone or in combination. A mouse model of a chemical carcinogen-induced lung tumor. Mice (A / J strain) are given 4 intraperitoneal injections (once a week) of tobacco and environmentally derived carcinogen BaP at a dose of 1 mg / Kg. Mouse flaxseed or lignan diet is initiated upon exposure. Mice are evaluated at various times after exposure to determine tumor burden, mouse weight, and overall health profile. Flaxseed reduces tumor burden in mice: the overall pathological profile of mouse lungs exposed to carcinogens. Representative clinical image of mouse lungs months after BaP exposure and edible flaxseed administration. Flaxseed reduces tumor burden in mice: histopathological profile of mouse lungs exposed to carcinogens. Representative H & E-stained lung sections of mouse lungs months after BaP exposure and edible flaxseed administration. The nodule indicated by the arrow in mice fed a control diet (upper panel) or flaxseed (lower panel) appears to be smaller in mice fed flaxseed. Each panel represents a different animal. A-B. Flaxseed reduces tumor burden in mice: quantitative assessment of tumor burden. Histological mouse lung sections were morphologically evaluated using image analysis software for overall tumor area (A) and nodule size (B). There was a significant decrease in the area of the lungs occupied by tumors in mice fed the flaxseed diet (p <0.03). Similarly, there was a trend for smaller tumor nodule sizes. A-B. Flaxseed reduces tumor burden in mice: quantitative assessment of tumor burden. Histological mouse lung sections were morphologically evaluated using image analysis software for the total number of tumor nodules per lung (A) and% tumor infiltrating the lung (B). There was a trend of less tumor nodules per lung (A) and less tumor infiltration (B) with flaxseed supplementation. Flaxseed supplementation prevents the wasting effect of lung cancer induced by BaP. Animal weights were measured over a period of 200 days after BaP exposure. Mice fed flaxseed diet and exposed to BaP showed higher weight than mice fed control diet and exposed to BaP. Experimental scheme for Example 5. Mammalian lignan metabolites can be detected in blood 4 days after daily intake of flaxseed (FS) and flaxseed lignan component (FLC) supplemented diets. In particular, enterodiol (ED) and enterolactone (EL) can be detected using liquid chromatography, tandem mass spectrometry (LC / MS / MS). The diet was designed to deliver comparable lignan levels, reflecting the lignan metabolite levels detectable in the two diets. Flaxseed (FS) and flaxseed lignan component (FLC) administered prior to asbestos exposure are intraperitoneal asbestos as evidenced by the number of macrophages (MF), neutrophils (PMN) and lymphocytes (Ly). Reduced abdominal inflammation induced by asbestos injection. In particular, FS and FLC significantly reduced macrophage influx in the abdomen. * P <0.05. A-D. Mice began FS and FLC diets and were exposed to asbestos 24 hours later. According to the experimental scheme of FIG. 34, 3 days after exposure, plasma (B, D) and abdominal (A, C) cytokine levels (TNFα and IL-1β) were determined using ELISA. Both diets showed a tendency to prevent secretion of pro-inflammatory cytokines in the abdomen and systemic circulation induced by asbestos exposure. A-B. Inflammatory cells also showed a tendency to decline with diet (A), but TNFα (B) and IL-1β (C) cytokine levels induced by 400 and 800 mg of asbestos asbestos were 1 day after asbestos exposure. Was significantly reduced by FLC added in the diet of (* p <0.05). * P indicates significance relative to control-fed mice exposed to asbestos. Inflammatory cells also showed a tendency to decline with diet (A), but TNFα (B) and IL-1β (C) cytokine levels induced by 400 and 800 mg of asbestos asbestos were 1 day after asbestos exposure. Was significantly reduced by FLC added in the diet of (* p <0.05). * P indicates significance relative to control-fed mice exposed to asbestos. Plasma concentrations of SDG and metabolites following oral gavage of various concentrations of SDG in mice. Antioxidant gene expression levels in the lung after oral gavage administration of various doses of SDG in mice. 2 shows the kinetics of plasma (A) and lung tissue (B) SDG levels and corresponding levels of AOE gene expression (C) after oral gavage of 100 mg / kg SDG in mice. Experimental scheme for Example 7. Experimental scheme for Example 7. Clinical trial design for Example 7. Western blot showing increased HO-1 and NQO-1 with 10% FS feeding in mouse nasal epithelium. Kinetics of HO-1 gene expression in human buccal epithelial cells after 40 gFS diet (* p <0.05 from day 0). Dynamics of urinary IsoP levels in one patient with FS intake. Kinetics of urinary 8-oxo-dGuo levels in healthy individuals with FS intake and lung transplant patients. SDG or flaxseed diet is hypothesized to reduce asbestos-induced ROS / inflammation. Experimental design (schematic diagram) of cellular asbestos exposure to detect inflammatory cytokine secretion and nitrosation / oxidative stress. SDG reduces asbestos-induced ROS secretion by human mesothelial cells in vitro. Assessment of asbestos-induced oxidative stress (ROS release) in cultured untreated macrophages: Asbestos-induced ROS was generated immediately after asbestos exposure and persisted during the observation period. A-B. SDG administered to macrophages hours after exposure to asbestos reduces oxidative stress. A-B. SDG administered to macrophages after several hours of exposure to asbestos reduces nitrosation stress. A-B. SDG administered to macrophages hours after exposure to asbestos reduces inflammatory cytokine (IL-1β) secretion. A-B. SDG administered to macrophages hours after exposure to asbestos reduces inflammatory cytokine secretion (TNF-α). Testing SDG in Asbestos-Induced Mesothelioma Using Two Mouse Models: Using at least two mouse models that are genetically susceptible to mesothelioma after asbestos exposure, we have tested against a single dose of asbestos in mice To assess the acute effects of flaxseed and SDG. We will also test whether flaxseed and SDG inhibit tumor development in a genetic model of asbestos-induced rapid progression MM. An SDG-rich (35% SDG) FLC diet given to MEXTAG mice exposed to asbestos reduced inflammation. Experimental design for asbestos exposure and flaxseed / SDG lignan blend evaluation in NF2 mice. A-B. Abdominal inflammation kinetics in NF2 mice after asbestos exposure: Influx cell influx peaked by 3 days after asbestos exposure and gradually decreased by 9 days. Therefore, in all subsequent experiments, 3 days were chosen as the inflammation assessment time point. A-B. Flaxseed and its SDG-rich lignan component reduced asbestos-induced inflammation (younger mice): Total leukocytes (A) decreased with dietary FS or FLC addition, but were not significant. However, looking at differences between cells, especially macrophage levels, levels were significantly reduced by both flaxseed and SDG lignan diets (B). A-C. Flaxseed and its SDG-rich lignan component reduced asbestos-induced inflammation (older mice): Older mice (A) exposed to abdominal asbestos were 3 compared to just 300,000 cells / mL It is more sensitive to asbestos as it shows 1,000,000 WBC / mL abdominal wash (10 times more). The results showed that neutrophils (B) and macrophages (C) of inflammatory cells were both significantly more in older mice than in young mice. A-B. SDG-rich flaxseed lignan extract (administered in the diet formulation) reduces asbestos inflammation in aged mice: male NF2 (129SV) (+/−) mice were injected with 400 μg asbestos on day 0 (Intraperitoneal). Mice were started on the test diet (0% FS or 10% FLC) in the week prior to asbestos exposure (day -7) and sacrificed on day 3 after asbestos exposure. Abdominal lavage (AL) was performed with 5 mL 1 × PBS (1 mL of abdominal lavage fluid was centrifuged and the supernatant was frozen). Plasma was collected and frozen at -80 ° C. Cells in the lavage fluid were evaluated and showed that a diet rich in SDG significantly reduced total WBC and neutrophils, macrophages and eosinophils. A-C. SDG-rich flaxseed lignan extract (administered in dietary formula) reduces asbestos inflammatory cytokine secretion and nitrosative stress in aged mice: male NF2 (129SV) (+/−) mice received 400 μg asbestos Injection on day 0 (intraperitoneal). Mice were started on the test diet (0% FS or 10% FLC) in the week prior to asbestos exposure (day 7) and sacrificed on day 3 after asbestos exposure. Abdominal lavage (AL) was performed with 5 mL 1 × PBS (1 mL of abdominal lavage fluid was centrifuged and the supernatant was frozen). Plasma was collected and frozen at -80 ° C. Cytokines IL1β and TNFα, and nitrite levels were significantly reduced by a diet rich in SDG.

  In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, those skilled in the art will appreciate that the invention may be practiced without these specific details. In other instances, well-known methods, operations and elements have not been described in detail so as not to obscure the present invention.

  Provided herein are therapeutic and prophylactic methods for radioprotection and chemoprotection using flaxseed, its bioactive ingredients, or its metabolites. In an exemplary embodiment, the bioactive ingredient includes secoisolaricylesinol diglucoside (SDG), secoisolarisiresinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, and the like. Isomers (including stereoisomers), or combinations thereof.

  The inventors of the present application have found that flaxseed, its physiologically active ingredients, and / or its degradation products or metabolites may cause radiation damage, hypochlorite ion induced damage, carcinogen induction of biomolecules, cells and tissues. It has been found to be effective in protecting against sexual injury and malignant lesions. Accordingly, the inventors have found that flaxseed, its bioactive ingredients, or its metabolites can cause biomolecules, cells, and tissues from being damaged by radiation, hypochlorite induced damage, carcinogenic damage and cancer development. Discovered that it can be used to protect.

  Subjects in need of radiation protection or radiation mitigation according to the methods provided herein will be exposed to, exposed to, or have been exposed to potentially harmful amounts of radiation It is. It will be appreciated that such exposure may be a single exposure to radiation, periodic exposure, sporadic exposure or continuous exposure. It will also be understood that such radiation exposure includes accidental, incidental or intentional exposure.

  Examples of subjects that may require radiation protection or radiation mitigation according to the methods of the present invention include patients exposed to radiation as part of therapy (eg, cancer patients in need of radiation therapy), diseases Or includes, but is not limited to, subjects exposed to radiation to diagnose the condition (eg, subjects receiving dental or bone x-rays, patients receiving PET scans, CT scans, etc.). Examples of subjects that may require radiation protection 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 occupational or lifestyle choice (eg, average Airplane flight crew or other high-frequency aircraft users, as well as space travelers; laboratory technicians and other workers; or exposure to use of electronic equipment that are exposed to levels higher than radiation levels Or persons exposed to radon accumulation (eg, residential or mine accumulation) or outdoor workers exposed to natural radiation from sunlight or persons exposed to sunlight. Other subjects that may require radiation protection according to the methods of the present invention are accidentally exposed to radiation such as leaks or releases (eg, reactor leaks or accidents or laboratory releases) Includes people. Also intended are those exposed to radiation as a result of a nuclear warhead explosion as a result of war or terrorism. Additional subjects include those exposed to terrorist blasts of traditional explosives that disperse radioactive material.

  Subjects in need of chemoprotection according to the methods provided herein will be exposed to, or are exposed to, potentially harmful amounts of carcinogens or other toxicants It is a subject. Such exposure may be a single exposure, a periodic exposure, a sporadic exposure, or a continuous exposure to one or several synthetic or natural carcinogens or other toxicants, e.g. combinations of chemical warfare agents. It will be appreciated. It will also be understood that such exposure includes accidental, incidental or intentional exposure. It will also be appreciated that such exposure may be direct exposure or indirect exposure. For example, indirect exposure to hypochlorite ions can be the result of direct exposure to ionizing radiation.

  Examples of subjects that may require chemical defense according to the methods of the present invention include those who are exposed to carcinogens or other poisons as a result of their occupational or lifestyle choice (eg, oil industry work , Toll officers, laboratory technicians and other workers who are exposed to vehicle exhaust particles). Other subjects that may require chemical protection by the methods of the present invention include carcinogenicity, such as leakage or release of carcinogenic substances (eg, asbestos, polycyclic aromatic hydrocarbons) in drinking water or air. Includes people who are accidentally exposed to the substance. People who are exposed to carcinogenic substances as a result of addiction (smokers) are also intended. Additional subjects include those exposed to the acts of terrorists who disperse carcinogens such as chemical warfare agents and other cancer-promoting substances.

  In one aspect, the invention relates to a method of protecting a biomolecule (such as genetic material such as a nucleic acid, protein or lipid), cell, or tissue from radiation damage in a subject in need thereof, the method comprising: Administering to a subject an effective amount of flaxseed, its bioactive ingredients, its degradation products or metabolites. Administration to the subject includes administration before, during and after exposure to harmful radiation exposure. The time before, during and after exposure can be seconds, minutes, hours, days, weeks, months or even years. The bioactive component includes secoisolaricillinol diglucoside (SDG), secoisolaricillinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or These combinations are included.

  In another aspect, the invention relates to a method of treating or preventing radiation damage in a subject that has been or will be exposed to radiation, said method comprising an effective amount of at least one of said subject Administration of a physiologically active ingredient, wherein the physiologically active ingredient is secoisolarisiresinol diglucoside (SDG), secoisolarisiresinol (SECO), enterodiol (ED), enterolactone (EL), these Including analogs, these stereoisomers, or combinations thereof.

  In another aspect, the invention protects biomolecules (such as genetic material such as nucleic acids, proteins or lipids), cells, or tissues in a subject in need thereof from radiation damage resulting from accidental radiation exposure. With respect to the method, the method comprises administering to the subject an effective amount of flaxseed, its bioactive component, its degradation product or metabolite.

  In another aspect, the invention relates to a method of protecting biomolecules (such as genetic material such as nucleic acids, proteins or lipids), cells, or tissues from radiation damage that causes aging.

  In another aspect, the present invention applies biomolecules (such as genetic material such as nucleic acids, proteins or lipids), cells, or tissues to both chemical and carcinogens and toxicants, both natural and synthetic, including chemical warfare agents. It relates to a method for protecting against radiation damage resulting from exposure.

  In another aspect, the invention relates to radiation damage resulting from radiation therapy for cancer treatment of biomolecules (such as genetic material such as nucleic acids, proteins or lipids), cells, or tissues in a subject in need thereof. The method includes administering to the subject an effective amount of flaxseed, its bioactive component, its degradation product or metabolite.

  In another aspect, the invention relates to a method of protecting a biomolecule (such as genetic material such as a nucleic acid, protein or lipid), cell or tissue in a subject in need thereof from radiation damage. Administering an effective amount of at least one bioactive ingredient to the subject, wherein the bioactive ingredient comprises secoisolarisiresinol diglucoside (SDG), secoisolarisiresinol (SECO), enterodiol (ED). ), Enterolactone (EL), analogs thereof, stereoisomers thereof, or combinations thereof. In some embodiments, the radiation damage results from accidental radiation exposure. In some embodiments, the radiation damage results from radiation therapy for cancer (eg, lung cancer) treatment.

  In another aspect, the present invention relates to a method for preventing radiation-induced damage to a biomolecule (such as genetic material such as nucleic acids, proteins or lipids), cells, or tissues in a subject in need thereof. Includes administering to the subject an effective amount of flaxseed, a physiologically active ingredient thereof, or a metabolite thereof.

  In another aspect, the present invention relates to a method for preventing radiation-induced damage to a biomolecule (such as genetic material such as nucleic acids, proteins or lipids), cells, or tissues in a subject in need thereof. Comprises administering to the subject an effective amount of at least one physiologically active ingredient, wherein the physiologically active ingredient is secoisolarisiresinol diglucoside (SDG), secoisolarisiresinol (SECO), enterodiol ( ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or combinations thereof.

  In another aspect, the invention relates to a method of protecting a biomolecule (such as genetic material such as a nucleic acid, protein or lipid), cell, or tissue from radiation damage in the cell, said method comprising: Contact with an amount of at least one bioactive component, wherein the bioactive component is secoisolaricillinol diglucoside (SDG), secoisolaricillinol (SECO), enterodiol (ED), enterolactone ( EL), analogs thereof, stereoisomers thereof, or combinations thereof.

  In another aspect, the invention relates to a method of protecting a biomolecule (such as genetic material such as a nucleic acid, protein or lipid), cell, or tissue in a subject in need thereof from damage by a carcinogen, This method comprises administering to the subject an effective amount of flaxseed, a bioactive component thereof, a degradation product or a metabolite thereof. Administration to the subject includes administration before, during and after exposure to harmful exposure to both natural and synthetic chemical carcinogens and toxicants. The time before, during and after exposure can be seconds, minutes, hours, days, weeks, months or even years. The bioactive component includes secoisolaricillinol diglucoside (SDG), secoisolaricillinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or These combinations are included.

  In another aspect, the invention relates to a method of protecting a biomolecule in a subject in need thereof from damage by carcinogens resulting from accidental exposure to both natural and synthetic chemical carcinogens and toxicants. The method includes administering to the subject an effective amount of flaxseed, a bioactive component thereof, a degradation product or a metabolite thereof.

  In another aspect, the invention relates to biomolecules (such as genetic material such as nucleic acids, proteins or lipids), cells, or tissues, both natural and synthetic chemical carcinogens that cause lung cancer or mesothelioma and The present invention relates to a method for protecting against damage from poisons.

  In another aspect, the invention relates to a method of protecting a biomolecule (such as genetic material such as a nucleic acid, protein or lipid), cell, or tissue from damage by a carcinogen, the method comprising: Contacting the cell or tissue with an effective amount of a physiologically active ingredient. Contact with the biomolecule, cell, or tissue includes contact before, during, and after damaging exposure to both natural and synthetic chemical carcinogens and toxicants. The time before, during and after exposure can be seconds, minutes, hours, days, weeks, months or even years. The bioactive component includes secoisolaricillinol diglucoside (SDG), secoisolaricillinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or These combinations are included.

  In another aspect, the invention provides a carcinogen-induced injury caused by a carcinogen-induced cancer in a subject that has been or will be exposed to one or more carcinogens. Relates to a method for treating or preventing malignant transformation or cancer development, the method comprising administering to the subject an effective amount of at least one bioactive component, wherein the bioactive component is secoisolarisiresinol diglucoside. (SDG), secoisolaricillinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or combinations thereof.

  In another aspect, the invention relates to a method of protecting a subject exposed to one or more carcinogens from a carcinogen-induced cancer, wherein the method comprises an effective amount of flaxseed, its physiological Administration of the active ingredient, its metabolites.

  In another aspect, the present invention provides a method of protecting a biomolecule (such as genetic material such as a nucleic acid, protein or lipid), cell, or tissue in a subject in need thereof from damage by hypochlorite ions. In this regard, this method comprises administering to the subject an effective amount of flaxseed, a bioactive component thereof, a degradation product or a metabolite thereof. Administration to the subject includes administration before, during and after exposure to harmful exposure to both natural and synthetic chemical carcinogens and toxicants. The time before, during and after exposure can be seconds, minutes, hours, days, weeks, months or even years. The bioactive component includes secoisolaricillinol diglucoside (SDG), secoisolaricillinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or These combinations are included.

  In another aspect, the invention relates to a method of treating or preventing hypochlorite ion-induced damage in a subject that has been or will be exposed to hypochlorite ion, the method comprising: Administering an effective amount of at least one physiologically active ingredient to the subject, wherein the physiologically active ingredient comprises secoisolarisiresinol diglucoside (SDG), secoisolarisiresinol (SECO), enterodiol (ED ), Enterolactone (EL), analogs thereof, stereoisomers thereof, or combinations thereof.

  In another aspect, the invention relates to a method of protecting biomolecules (such as genetic material such as nucleic acids, proteins or lipids), cells, or tissues from damage by hypochlorite ions, the method comprising: Contacting the biomolecule, cell, or tissue exposed to or expected to be exposed to chlorate ions with an effective amount of a bioactive component. Contact with the biomolecule, cell, or tissue includes contact with both natural and synthetic chemical carcinogens and toxicants prior to, during and after exposure to harmful exposure. The time before, during and after exposure can be seconds, minutes, hours, days, weeks, months or even years. The bioactive component includes secoisolaricillinol diglucoside (SDG), secoisolaricillinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or These combinations are included.

  In another aspect, the invention relates to a composition for use in one of the aforementioned methods.

  Flaxseed, its biologically active ingredients, and metabolites thereof are known in the art and are described in US Patent Application Publication Nos. 2010/0239696, 2011/0300247, and 2014/0308379, and International Publication No. 2014/200964. Each of these published patents is incorporated herein by reference in its entirety.

  The major lignan found in flaxseed is 2,3-bis (3-methoxy-4-hydroxybenzyl) butane-1,4-diol (secoisolariciresinol or SECO), which is naturally found in plants. In this state, the conjugate is stored as secoisolariciresinol diglucoside (SDG). SDG is metabolized into enterodiol (ED) and enterolactone (EL) in the human intestine. Synthetic analogs of enterodiol and enterolactone are known (see, for example, Eklund et al., Org. Lett., 2003, 5: 491).

  A “degradation product” is the product of the breakdown of a molecule such as SDG into smaller molecules.

  A “metabolite” is a substance produced by metabolism or a metabolic process. For example, the metabolite of SDG is EL or ED.

  One skilled in the art will appreciate that the metabolite may be a chemically synthesized equivalent of the natural metabolite.

  An “analog” is a compound whose structure is related to the structure of another compound. The analog may be a synthetic analog.

  A “component” or “component” is an element or component in a mixture or compound.

  A “product” is a substance resulting from a chemical reaction.

  An “extract” is a preparation containing the active ingredient or concentrated essence of a substance, for example derived from flaxseed.

  A “pharmaceutical composition” is an effective amount of an active ingredient, eg, (S, S) -SDG, (R, R) -SDG, meso SDG, SDG, SECO, with a pharmaceutically acceptable carrier or diluent. , EL, ED and analogs thereof. “Therapeutically effective amount” refers to that amount which produces a therapeutic effect for a given condition and administration regimen.

  The compositions described herein may comprise a “therapeutically effective amount”. “Therapeutically effective amount” refers to an amount effective to achieve the desired therapeutic result at the required dosage and for the required period of time. A therapeutically effective amount can vary depending on factors such as the condition, age, sex, and individual weight, and the ability of the composition to elicit a desired response in the individual. A therapeutically effective amount is also such that the therapeutically beneficial effect exceeds the toxic or harmful effects of the molecule.

  As used herein, the phrase “pharmaceutically acceptable” refers to a compound, substance, composition, carrier, and / or dosage form that is within the scope of sound medical judgment. Suitable for use in contact with human and animal tissues, without excessive toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable benefit risk ratio.

  “Pharmaceutically acceptable excipients” are excipients that are useful in the preparation of pharmaceutical compositions that are generally safe, non-toxic, and not biologically or otherwise inappropriate And includes excipients that are acceptable for veterinary use and pharmaceutical use in humans. As used herein, “pharmaceutically acceptable excipient” includes one or more such excipients.

  The pharmaceutical composition is available to those skilled in the art, such as oral, parenteral, transmucosal, transdermal, intramuscular, intravenous, intradermal, subcutaneous, intraperitoneal, intraventricular, intracranial, intravaginal, intratumoral, or oral cavity. Administration to the subject can be by any suitable method known. Controlled release can also be used by embedding the active ingredient in a suitable polymer and then inserting it subcutaneously, intratumorally, intraorally, as a patch on the skin, or inserted into the vagina. Also included is coating a medical device with an active ingredient.

  In some embodiments, the pharmaceutical composition is administered orally and is therefore formulated in a form suitable for oral administration, ie, a solid or liquid formulation. 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. In some embodiments, the active ingredient is formulated in a capsule. In this embodiment, the composition of the invention comprises a desiccant in addition to the active compound and inert carrier or diluent, in addition to other excipients and gelatin capsules.

  In some embodiments, the pharmaceutical composition is administered by intravenous, intraarterial, or intramuscular injection of the liquid formulation. In some embodiments, the pharmaceutical composition is a liquid formulation formulated for oral administration. In some embodiments, the pharmaceutical composition is a liquid formulation formulated for intravaginal administration. Suitable liquid formulations include solutions, suspensions, dispersions, emulsions, oils and the like. In some embodiments, the pharmaceutical composition is administered intravenously and is thus formulated in a form suitable for intravenous administration. In another embodiment, the pharmaceutical composition is administered intraarterially and is therefore formulated in a form suitable for intraarterial administration. In some embodiments, the pharmaceutical composition is administered intramuscularly and is therefore formulated in a form suitable for intramuscular administration. In some embodiments, the pharmaceutical composition is administered orally and is thus formulated in a form suitable for oral administration.

  In some embodiments, the pharmaceutical composition is administered topically to the body surface and is thus formulated in a form suitable for topical administration. Suitable topical formulations include gels, ointments, creams, lotions, drops, controlled release polymers and the like. For topical administration, flaxseed, its bioactive ingredients, its metabolites are prepared and as a solution, suspension, or emulsion in a physiologically acceptable diluent, with or without a pharmaceutical carrier Applied.

  In some embodiments, the pharmaceutical compositions provided herein are controlled release compositions, i.e., compositions in which flaxseed, its bioactive ingredients, its metabolites are released over a period of time after administration. is there. Controlled or sustained release compositions include formulations in lipophilic stores (eg, fatty acids, waxes, oils). In other embodiments, the composition is an immediate-acting composition, ie, a composition in which all flaxseed, its bioactive ingredients, and its metabolites are released immediately after administration.

  In some embodiments, the composition for use in the methods provided herein is administered in a therapeutic amount once daily. In some embodiments, the composition is administered once every two days, twice a week, once a week, or once every two weeks.

  Techniques for extracting and purifying SDG are known in the art and are described in US Pat. No. 5,705,618. This patent is incorporated herein by reference. Techniques for synthesizing SDG, its stereoisomers and analogs are described in Misra OP, et al. , Bioorganic & Medicinal Chemistry Letters 2013, (19): 5325-5328 and WO 2014/200964. These documents are incorporated herein by reference in their entirety. The bioactive ingredients for use in the methods provided herein are also enterodiol (ED) or enterolactone in mammalian, readily metabolizable forms, as is known in the art. It can also be chemically synthesized directly into (EL).

  (S, S) -SDG, (R, R) -SDG, (S, R) -SDG, (R, S) -SDG, meso SDG, SECO, EL, ED or analogs thereof are 0.1 ng / Kg to 500 mg / kg can be administered.

  Treatment with (S, S) -SDG, (R, R) -SDG, (S, R) -SDG, (R, S) -SDG, meso SDG, SDG, SECO, EL, ED or analogs thereof Ranging from a single dose to days, months, years, or indefinite periods.

  As used herein, “treatment” refers to therapeutic treatment or prophylactic or preventative treatment, the purpose of which is to prevent or alleviate a targeted disease state or disorder as described herein. , Or both. Thus, a composition for use in the methods provided herein can be administered to a subject prior to exposure to, for example, radiation, carcinogens, toxicants, or hypochlorite ions. In some cases, a composition for use in the methods provided herein can be administered to a subject after exposure. Accordingly, treating a condition described herein can refer to preventing, inhibiting, or suppressing the condition in a subject.

  Further, as used herein, the terms “treat” and “treatment” refer to therapeutic treatment, and prophylactic or preventative treatment, the purpose of which is unfavorable physiological associated with the disease or condition. To prevent or slow down (reduce) major changes. Beneficial or desirable clinical outcomes, whether detectable or not, alleviate symptoms, reduce the degree of the disease or condition, stabilize the disease or condition (ie, the condition in which the disease or condition does not worsen) ), Slowing or slowing the progression of the disease or condition, ameliorating or alleviating the disease or condition, and remission (partial or complete) of the disease or condition. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. People in need of treatment include, for example, people who have already been exposed to radiation, carcinogens, toxicants, or hypochlorite ions, as well as those who are or are expected to be exposed Is included.

  In some embodiments, subjects in need of the treatments and methods and compositions described herein include patients with lung diseases and disorders such as asthma, cancer, COPD, and mesothelioma. However, it is not limited to these. In some embodiments, suitable subjects include patients with disorders and conditions related to aging such as cardiovascular disorders and conditions, skin sagging and central nervous system (CNS) diseases (eg, Alzheimer's dementia). Can do. In some embodiments, suitable subjects may include patients with skin disorders and conditions (eg, psoriasis), and patients with cosmetic skin conditions (eg, wrinkles and stains). In some embodiments, suitable subjects may include gastrointestinal disorders and conditions, such as patients with IBD and Crohn's disease. In some embodiments, suitable subjects may include patients with cardiovascular disorders and conditions. In some embodiments, suitable subjects may include patients with melanoma. In some embodiments, suitable subjects may include patients with eye diseases such as macular degeneration. In some embodiments, suitable subjects may include patients with cancer, eg, breast cancer, prostate cancer and uterine cancer. In some embodiments, suitable subjects may include patients with cognitive impairment and other cognitive impairments.

  The term “subject” refers to mammals such as humans, companion animals (eg, dogs, cats, birds, etc.), livestock (eg, cows, sheep, pigs, horses, poultry, etc.) and laboratory animals (eg, rats, Mouse, guinea pig, bird, etc.). In addition to humans, subjects can be dogs, cats, pigs, cows, sheep, goats, horses, buffalo, ostriches, guinea pigs, rats, mice, birds (eg parakeets) and other wild, domesticated or Commercially useful animals (eg, chickens, geese, turkeys, fish) may be included. The term “subject” does not exclude individuals who are normal in all respects. The term “subject” includes, but is not limited to, a human in need of treatment for or susceptible to a condition or its sequelae.

  The terms “about” and “approximate” mean within an acceptable error range for a particular value as determined by one skilled in the art. This acceptable error range may depend in part on how the value is measured or determined, i.e. the constraints of the measurement system.

  The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. However, these examples should not be construed as limiting the broad scope of the invention in any way.

Example Example 1
Synthetic (S, S) and (R, R) -secoisolarisiresinol diglucoside (SDG) protects naked plasmid DNA and genomic DNA from gamma radiation damage.
As a result of nuclear decay, three types of radiation are generated: alpha (α) rays with positive charges, beta (β) rays with negative charges, and gamma (γ) rays that do not retain charge.
In the case of gamma rays, electromagnetic waves have a very small wavelength (<0.005 nm) and thus have high energy that can ionize molecules and atoms. In biological systems or in solution, ionizing radiation generates hydroxyl radicals (.OH) by radiolysis of water. These hydroxyl radicals (.OH) are a major source of ionizing radiation-induced damage to cellular components such as lipids, proteins and genomic DNA. Hydroxyl radicals (.OH) generated by gamma rays cause single and double strand breaks in DNA. (.OH) radicals damage DNA by removing H atoms from deoxyribose and purines and pyrimidine bases, or by adding to double bonds in bases. These reactions result in DNA strand breaks.

Compounds with antioxidant and free radical scavenging properties can function as radioprotectants and prevent radiation-induced DNA damage. In order to isolate SDG from natural resources in connection with the high cost, variability and difficulty of producing large quantities of secoisolarisiresinol diglucoside (SDG) enabling preclinical and clinical trials SDG was chemically synthesized because of the complexity of the extraction, purification and concentration methods. The natural compounds vanillin and glucose were used to successfully synthesize two enantiomers of SDG (these structures are shown below): SDG (S, S) and SDG (R, R) (Mishra). et al., Bioorganic & Medicinal Chemistry Letters 2013, (19): 5325).

  Many studies by Christofidou-Soromidou et al. And other researchers have shown that SDG is a powerful antioxidant and a powerful free radical scavenger. Importantly, recent studies have shown that synthetic SDG enantiomers have potent antioxidant and free radical scavenging properties (Mishra et al., Bioorganic & Medicinal Chemistry Letters 2013, (19): 5325-5328). In this example, the radioprotective properties of the synthesized SDG enantiomers (S, S) -SDG and (R, R) -SDG were investigated and evaluated compared to commercially available SDG. Using a plasmid DNA relaxation assay to measure the ability of SDG to prevent the conversion of plasmid DNA (pBR322) from supercoil to open coil after exposure of the plasmid to gamma irradiation, as well as to gamma irradiation of DNA The radioprotective properties of the three compounds were evaluated by evaluating the inhibition of genomic DNA fragmentation after exposure. SDG is metabolized by intestinal bacteria to produce secoisolarisiresinol (SECO), enterodiol (ED) and enterolactone (EL). Therefore, the effect of these SDG metabolites on gamma irradiation induced fragmentation of genomic DNA was also evaluated.

Materials and Methods Chemicals Plasmid DNA (pBR322), ethidium bromide, ultra high purity 10X TAE buffer and 1 kb DNA ladder were purchased from Invitrogen (Life Technologies, Carlsbad, CA). Agarose (ultra high purity) and calf thymus DNA were purchased from Sigma-Aldrich (St. Louis, MO). Secoisolarisiresinol diglucoside (SDG) (commercially available), secoisolarisiresinol (SECO), enterodiol (ED) and enterolactone (EL) were purchased from Chromadex (Irvine, CA).

Synthesis of Secoisolariciresinol Diglucoside (SDG) Synthetic SDG (R, R) and SDG (S, S) stereoisomers were prepared according to the method of Misra et al. , Bioorganic & Medicinal Chemistry Letters 2013, (19): 5325-5328.

Exposure of plasmid DNA and calf thymus DNA to gamma radiation Plasmid DNA (pBR322) or pups with or without various concentrations of SDG (R, R), SDG (S, S) and SDG (commercially available) Bovine thymus DNA samples were transferred to 1.7 Gy / pH in phosphate buffered saline (PBS) at pH 7.4 using a Mark1 cesium (Cs-137) irradiator (JL Shepherd, San Fernando, Calif.). Exposed to gamma rays at a dose rate of minutes.

Measurement of radiation-induced plasmid DNA relaxation The effect of test compounds on radiation-induced strand breaks and supercoil (SC) to open coil (OC) conversion was determined using plasmid DNA (pBR322) (Life Technologies, Carlsbad, CA). Measured using. Plasmid DNA (500 ng) in PBS (pH 7.4) was mixed with various concentrations (25-250 μM) of SDG (R, R), SDG (S, S) and SDG (commercially available) at 25 Gy in PBS. Irradiated. Thirty minutes after radiation exposure, the sample was mixed with a loading dye and subjected to agarose (1%) gel electrophoresis at 100 V in TAE buffer (pH 8.3). The gel was stained with ethidium bromide (0.5 μg / mL) for 40 minutes, washed for 20 minutes, and then visualized with a UV transilluminator (Bio-Rad, Hercules, Calif.). The captured gel images were scanned and the density of open coil (OC) and supercoil (SC) plasmid DNA bands was determined by the Gel-doc image analyzer program. SC and OC plasmid DNA concentrations were expressed as% of the total density (OC + SC).

Measurement of radiation-induced DNA fragmentation The effect of test compounds on radiation-induced strand breaks in DNA was measured using calf thymus DNA (Sigma, St. Louis, MO). DNA (500 ng) in PBS (pH 7.4) was mixed with various concentrations (25 to 250 μM) of SDG (R, R), SDG (S, S) and SDG (commercially available) and irradiated at 50 Gy for 30 minutes . The second series of experiments was performed with varying concentrations in the range of 0.5-10 μM. Samples were mixed with a loading dye and subjected to agarose (1%) gel electrophoresis at 100 V in TAE buffer (pH 8.3). The gel was stained with ethidium bromide (0.5 μg / mL) for 40 minutes, washed for 20 minutes, and then visualized with a UV transilluminator (Bio-Rad, Hercules, Calif.). Incorporated gel images were scanned and the density of calf thymus DNA fragments was determined by the Gel-Pro image analyzer program (Media Cybernetics, Silver Spring, MD). The density of the low molecular weight (<6,000 bp) and high molecular weight (> 6,000 bp) fragments of calf thymus DNA was expressed as a percentage of the total density (low molecular weight + high molecular weight).

Data analysis Data obtained is expressed as mean ± standard deviation. Data were subjected to a one-way analysis of variance (ANOVA) with post hoc comparison using Bonferroni's correction using the Statview Program. A p value ≦ 0.05 was considered significant.

Results Plasmid DNA (pBR322) was used to determine the radioprotective potential of synthetic SDG (R, R), SDG (S, S), and SDG (commercially available). The radioprotection assay used for this study is based on the principle that plasmid DNA after exposure to gamma rays migrates slower than unexposed plasmid DNA. This is simply due to the fact that supercoiled plasmid DNA moves faster in agarose gels due to its compact size. In comparison, radiation-induced breaks in plasmid DNA unwind the supercoil, resulting in a relatively larger size plasmid that moves at a slower rate in the gel. Thus, determining the density of open coil plasmid DNA compared to supercoiled plasmid DNA indicates the extent of radiation-induced damage.

Radiation results in dose-dependent SC-to-OC DNA plasmid conversion. In order to select a dose of radiation that will cause significant DNA damage, but still allow a therapeutic window to test radiation mitigants. DNA was exposed to 10, 25 and 50 Gy gamma rays. The results shown in FIG. 11A indicate that there is a radiation dose dependent increase in OC type plasmid DNA as well as a radiation dose dependent decrease in SC type. The distribution of SC and OC (FIG. 1B) is as follows: SC% is 0, 10, 25 and 50 Gy, from 68.73 ± 2.54% to 50.91 ± 2.31, 38.37 ± 3.73, respectively. And 35.66 ± 4.24% (p <0.05). At the same time, the OC% is 0, 10, 25 and 50 Gy from 31.26 ± 2.50% to 49.08 ± 2.31%, 61.62 ± 3.73% and 67.33 ± 4. It shows an increase to 24% (p <0.05). Based on these initial experiments, a radiation dose of 25 Gy (with this dose, a fairly clearly identifiable damage was achieved) was selected for subsequent experiments to measure the radiation protection properties of different SDGs. .

Radioprotective Activity of Synthetic SDG Using Plasmid DNA Relaxation Assay Exposing plasmid DNA to selected doses of 25 Gy gamma radiation (see FIG. 1), DNA inhibition (formation of OC from SC)% inhibition of various concentrations (25-25) 250 μM) for each SDG agent (synthetic and commercially available).

A representative gel blot of plasmid DNA after exposure to 25 Gy in the presence of 25, 50, 100 and 250 μM SDG (S, S) is shown in FIG. 2A, and semiquantitative concentration analysis is shown in FIG. 2B, compared to the control % Inhibition is shown in FIG. 2C. Interestingly, in the presence of increasing concentrations of SDG (S, S) (25, 50, 100 and 250 μM), the ratio of SC type increased and OC was significantly (p <0.05) and dose-dependent. The mold concentration decreased. Using the% inhibition plot (FIG. 2C), an EC ₅₀ value (ie, the effective concentration (EC) required to prevent 50% of plasmid relaxation at 25 Gy) can be determined for each drug and this value is In the case of SDG (S, S), it is 141.77 μM. This value for preventing plasmid DNA relaxation is equivalent to the EC ₅₀ value for removing DPPH free radicals. These results demonstrate the radioprotective properties of the synthetic SDG (S, S) enantiomer. Similar results are shown for the SDG (R, R) enantiomer (FIGS. 2D-F) and SDG (commercially available) (FIGS. 2G-I), with EC ₅₀ of 127.96 μM and 98.38 μM, respectively. These values for preventing plasmid DNA relaxation are equivalent to their respective EC ₅₀ values for removing DPPH free radicals. These results demonstrate the radioprotective properties of both synthetic and commercially available natural SDG.

Radiation causes dose-dependent DNA fragmentation ranging from high molecular weight to low molecular weight fragments. Radiation induces an increase in DNA fragmentation, as shown in the DNA gel in FIG. 3A. Based on size, calf thymus DNA fragments were divided into two groups: high molecular weight (> 6,000 bp) size and low molecular weight (<6,000 bp) size. The distribution of high and low molecular weight fragments (FIG. 3B) shows that the percentage of high molecular weight DNA is 88.16 ± 0.50%, 67.82 ± 7.89 and 34.94 ± 4.45% at 25 and 50 Gy. (P <0.05) indicates a decrease. At the same time, the proportion of low molecular weight fragments increased from 11.83 ± 0.50 to 32.17 ± 7.89% and 65.05 ± 4.45% (p <0.05) at 25 and 50 Gy, respectively. The results (FIG. 3B) show a significant decrease in high molecular weight DNA fragments and a significant increase in low molecular weight DNA fragments, indicating damage to DNA at 50 Gy. Based on these initial experiments, a radiation dose of 50 Gy (at which a clear and identifiable calf thymus DNA fragmentation was observed) was used for subsequent experiments to determine the radioprotective properties of different SDGs. Selected.

Radioprotective activity of synthetic SDG using calf thymus DNA fragmentation assay As described above, synthetic SDG (R, R), SDG (S, S) and SDG using radiation-induced fragmentation of calf thymus DNA. (Commercially available) radiation protection potential was determined.
High SDG concentration (25-250 μM): 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 μM SDG (S, S). In the presence of increasing concentrations of SDG (S, S) (25, 50, 100 and 250 μM), the proportion of high molecular weight DNA type increased significantly (p <0.05) after radiation exposure, but low molecular weight fragments Decreased. The distribution of high and low molecular weight DNA types in the presence of various concentrations of SDG (S, S) is shown in FIG. 4B. These results show the radioprotective properties of our synthetic SDG (S, S) enantiomer using calf thymus genomic DNA. Similarly, the results presented in FIGS. 4C-D and 4E-F show the radioprotective properties of synthetic SDG (R, R) and SDG (commercially available), respectively. These results show the radioprotective properties of synthetic SDG (R, R) and (S, S) enantiomers using calf thymus genomic DNA.

To further determine the lower limit of SDG in DNA protection, a series of DNA fragmentation experiments were performed that tested all three SDGs at lower concentrations in the range of 0.5-10 μM.
Low SDG concentration (0.5-10 μM): The results of experiments performed at low concentrations of SDG (S, S), SDG (R, R) and SDG (commercially available) A comparison with EC ₅₀ values is shown in FIG. Similar to higher SDG concentrations, these results shown in this section using the calf thymus DNA fragmentation assay show that even at low concentrations, synthetic SDG (S, S), and SDG (R, R) Demonstrate that the enantiomers have strong radioprotective properties.

Radioprotective activity of SDG metabolites using a calf thymus DNA fragmentation assay The radiation-induced fragmentation of calf thymus DNA described above was used to determine the radioprotective potential of SDG metabolites SECO, ED and EL, and SDG Compared with. Each test agent at a concentration of 10 μM was selected based on the previous findings shown above as the median effective dose. The results are shown in FIG. The data show that SDG and its metabolites SECO, ED, EL are equally effective in terms of their radioprotective properties.

Discussion The results of this example show that the synthetic SDG (S, S) and SDG (R, R) enantiomers have strong radioprotective properties. The radioprotective potential of these enantiomers, determined using plasmid DNA (pBR322), increased with increasing their concentration. These synthetic SDG (S, S) and SDG (R, R) enantiomers prevented radiation-induced damage to plasmid DNA in a concentration-dependent manner. The radioprotective potential of the synthetic isomers of SDG was comparable to commercial SDG. The synthetic enantiomers SDG (S, S) and SDG (R, R) also prevented radiation-induced DNA fragmentation of calf thymus genomic DNA. At the lowest concentrations tested, SDG (S, S) and SDG (R, R) completely prevent radiation-induced production of low molecular weight fragments of calf thymus DNA, and synthetic SDG (S, S) and SDG (R , R) showed strong radioprotective properties of the enantiomers. The results using low concentrations of SDG (S, S), SDG (R, R) and SDG (commercially available) show that the concentrations required to protect calf thymus DNA from gamma damage are their antioxidant and It was much lower than the EC ₅₀ value for free radical scavenging activity. Importantly, the SECO, ED and EL of mammalian lignan SDG metabolites showed equally strong DNA protective properties.

  Flavonoids have strong antioxidant activity. In particular, such polyphenols are known to have free radical scavenging activity and are more effective in vitro than vitamins E and C. Edible and medicinal plants with antioxidant properties are also known and useful radioprotectors, as they are known to prevent many human diseases associated with oxidative stress. Antioxidants such as vitamins and minerals reduced the level of chromosomal aberration inducers in Chernobyl workers after many years of radiation exposure. We have investigated the role of whole grain edible flaxseed, a grain rich in lignan polyphenols, and a SDG rich flaxseed lignan formulation in radiation-induced damage using a mouse model of chest radiation damage. We have shown that flaxseed mitigated radiation-induced inflammation and oxidative stress in mice when administered both before and after radiation exposure. We also showed that irradiated mice fed only with the lignan component of flaxseed enriched with lignan biphenol SDG showed significant improvements in hemodynamic measurements and survival, while at the same time demonstrating lung inflammation and tissue oxidative damage. It proved that it also showed improvement. These studies have shown that flaxseed is protective against the effects of lignan SDG against radiation-induced tissue damage in vivo.

Increased production of reactive oxygen species (ROS) such as superoxide anion (O ₂ ⁻ ), hydroxyl radical (.OH), and hydrogen peroxide leads to tissue damage under various experimental and pathological conditions. Reactive oxygen species cause cell damage by oxidative modification of cell membrane lipids, proteins and genomic DNA. Many studies have shown that extracted, purified, or synthetic flaxseed is a powerful antioxidant in vitro as well as in vivo. Thus, SDG as an antioxidant has therapeutic potential under a variety of experimental and disease states, including radiation-induced tissue damage in patients undergoing radiation therapy.

  Polyphenols generally exist as glycosides in plants and have antioxidant properties. Flavonoids, as antioxidants, interfere with the activity of enzymes involved in the production of reactive oxygen species, scavenge free radicals, chelate transition metals and render them redox inactive in the Fenton reaction. Secoisolarisiresinol (SDG) is the major lignan in flaxseed and has been shown to be a potent antioxidant in vitro as well as in vivo. In order to investigate the therapeutic potential of flaxseed lignansecoisoralisiresinol (SDG), SDG was synthesized by a chemical reaction using vanillin as a precursor molecule, and synthetic SDG (R, R) and SDG (S, S) Antioxidant properties were clarified by evaluating their reducing power, metal chelation potential, and free radical scavenging activity against hydroxyl, peroxy and DPPH radicals. In this example, we show the radioprotective properties of synthetic SDG (R, R), SDG (S, S) enantiomers and commercial SDG (as a control) against plasmid DNA (pBR322) and calf thymus DNA. It was investigated by evaluating their potential to prevent gamma irradiation-induced damage. Radiation-induced damage to plasmid DNA was assessed by an increase in open coil plasmid DNA and a decrease in supercoiled plasmid DNA. Radiation-induced damage to genomic DNA was assessed by determining the level of DNA fragmentation. In this example, we investigated the efficacy of synthetic SDG (R, R), SDG (S, S) and commercial SDG against radiation-induced DNA damage in a cell-free system.

  The antioxidant properties of SDG molecules have been shown previously. We have shown that natural commercial SDG has strong free radical scavenging properties in cells exposed to gamma radiation. The antioxidant and free radical scavenging properties of these synthetic SDG (R, R) and SDG (S, S) enantiomers were investigated, with strong reducing power, high metal ion chelating potential, and hydroxyl, peroxy And it showed high free radical scavenging activity against DPPH radical. These properties of synthetic SDG (R, R) and SDG (S, S) make these molecules powerful for modulating cellular redox states, reducing metal ion concentrations, and eliminating oxygen free radicals. Clarify showing potential. These properties of the synthetic SDG enantiomers suggest their ability to act at and inhibit all three steps of initiation, growth and termination of free radical reactions, It suggests that the mechanism may be involved in the radioprotective properties of SDG (S, S) and SDG (R, R) enantiomers in vivo.

There was one observation that maximum radioprotection of genomic DNA by SDG was already achieved at a concentration of about 5.0 μM, which is significantly less than the EC ₅₀ value for their free radical scavenging and antioxidant effects. Thus, SDG as an antioxidant and free radical scavenger can also function as a DNA radioprotectant and radiation mitigator.

  In summary, this example demonstrates that the synthetic SDG (S, S) and SDG (R, R) enantiomers have potent radioprotective properties. The radioprotective potential of these enantiomers was determined using plasmid DNA (pBR322) and calf thymus DNA. Synthetic SDG (S, S) and SDG (R, R) enantiomers prevented radiation-induced damage to plasmid DNA in a concentration-dependent manner. The synthetic enantiomers SDG (S, S) and SDG (R, R) also prevented radiation-induced fragmentation of calf thymus genomic DNA. At a concentration of 5 μM, SDG (S, S) and SDG (R, R) completely prevent radiation-induced production of low molecular weight fragments of calf thymus DNA, and the powerful radioprotective properties of these enantiomers. Show.

Example 2
Radioprotective properties of lignansecoisolariciresinol diglucoside (SDG) in lung cells Radiation injury to cells is initiated by the generation of reactive oxygen species (ROS). The extent of damage done by ROS to cellular mechanisms includes lipid peroxidation, DNA protein cross-linking, base modification, adduct formation and single and double strand breaks (DNA SSB and DSB). These modifications have been linked to radiation-induced apoptosis and cell death. Because cellular DNA damage is a known determinant in radiation-induced cell death, identify and exploit drugs that can protect DNA against radiation damage by preventing free radical reactions or by modulating radiation-induced apoptosis Great efforts have been made to achieve this.

  Prevention of radiation-induced genotoxicity can be achieved by the presence of antioxidants in the system upon exposure. Antioxidants can be ROS scavengers that interfere with free radical chain reactions, so that supplementation with antioxidants can protect cellular DNA from radiation-induced oxidative stress. Many synthetic and natural antioxidant compounds have been investigated for radioprotective efficacy. However, most of them exhibit intrinsic toxicity and side effects at their effective concentrations, or have a short shelf life and low bioavailability. Therefore, research on effective and non-toxic radioprotectors has led to investigations into edible antioxidants and dietary supplements.

  We evaluated the protective effects of edible flaxseed (FS) supplementation in preclinical mouse models of pulmonary oxidative damage such as hyperoxia, gastric acid inhalation injury, and ischemia / reperfusion injury. We conclude that the protective effect of FS may be due in part to its ability to increase antioxidant enzyme expression in lung tissue. Importantly, edible FS mitigated the adverse effects of chest radiation when administered both before and after exposure. In these studies, flaxseed reduced radiation-induced lung tissue damage, reduced lung inflammation, and prevented pulmonary fibrosis.

  Previous reports suggested that the effects of various flaxseed could be attributed to the flaxseed lignans that have been shown to have antioxidant, anti-inflammatory and anti-carcinogenic effects. Secoisolarisiresinol diglucoside (SDG) is the main FS lignan (about 1% of dry weight), which probably contributes to the beneficial health benefits of FS cereals. SDG is metabolized in the gut by intestinal bacteria to mammalian lignans, ie enterodiol (ED) and enterolactone (EL). SDG has been shown to be beneficial in the treatment of preclinical models of many diseases such as atherosclerosis and diabetes. SDG has also been reported to exert cardioprotective effects in animal models. FS lignans are protective against a variety of cancer types, as summarized in a recent review of the health benefits of SDG by Adolphe et al. (Br J Nutr 2010, 103: 929) and are also melanoma in animals. It has been reported to reduce metastasis.

  Furthermore, the antioxidant and free radical scavenging properties of SDG are well documented, which is most important because the free radical scavenging ability of a compound can be directly related to its radioprotective efficacy. In our investigation of pulmonary endothelial cells, SDG exhibits free radical scavenging properties when cells are exposed to γ-irradiation, while SDG-rich whole flaxseed lignan component (FLC) has been found to provide radiation protection and radiation in mice. Mediated mitigation. However, the characterization of the radiation protection properties of SDG has not been established.

  This study was conducted to clarify the radioprotective capacity of FS lignan SDG and to investigate possible mechanisms involved in its action. The primary objective of this study is to evaluate the role of SDG in radiation-induced clonogenic cell death in mouse primary lung cells, particularly epithelium, endothelial cells and fibroblasts. Since radiation-induced proliferative death of cells is directly related to cellular DNA damage, we determined whether SDG can protect cells from radiation-induced DNA strand breaks in alkaline comet assay (SSB) and γ-H2AX focus Was evaluated using the formation of (DSB). Furthermore, we investigated the effect of SDG pretreatment in preventing mouse primary lung cells from IR-induced cell death. Many studies have demonstrated the role of the pro-apoptotic protein Bax (Bcl-2 binding X protein) in IR-induced cell death. We also analyzed the direct effect of SDG on the expression of Bax and its antagonist Bcl-2 (B cell leukemia / lymphoma 2) mRNA, and the mechanism of SDG protection is a shift in the ratio of these important apoptosis regulators. Decided if relevant. Our findings reveal that lignan SDG, a potent bioactive component in FS, mediates radioprotection in lung cells, thus providing new insights into the radioprotective effects of FS.

Materials and Methods Reagents Secoisoralisiresinol diglucoside (SDG) is a commercial product (ChromaDex, Inc., CA). Comet assay kits are available from Trevigen, Inc. , (Gaithersburg, MD). P-Histone H2AX (rabbit mAb) is available from Cell Signaling Technology, Inc. , (Danvers, MA)). Phosphate buffered saline (PBS), bovine serum albumin (BSA), Dulbecco's modified Eagle medium (DMEM) (containing L-glutamine, glucose 1 g / l, sodium bicarbonate free), hepes buffer, bovine serum albumin ( BSA), ethylenediaminetetraacetic acid (EDTA), 4 ′, 6-diamino-2-phenylindole (DAPI), fetal bovine serum (FBS), collagenase, Triton X100 and dispase are available from Sigma-Aldrich, St. Purchased from Louis, MO, USA.

Cell lines Fibroblasts and endothelial cells were isolated from C57 / bl6 mice. For fibroblast isolation, mouse lungs were harvested, minced and incubated with dispase (2 mg / ml) for 45 minutes. Several pieces were plated and the fibroblasts were cultured and used between passages 3 and 10 as previously described. Pulmonary microvascular endothelial cells (PMVEC) were isolated from mouse lungs as previously described. Briefly, cells were isolated by treating freshly harvested mouse lungs with collagenase followed by attachment to mAb-coated magnetic beads against platelet endothelial cell adhesion molecule (PECAM). Epithelial cells (C10) are initially derived from normal BALB / c mouse lung explants, are non-tumorigenic, contact-inhibited, and have characteristics of type 2 alveolar cells at early passages.

Clonogenic survival Exponentially proliferating cells were seeded as single cells and incubated overnight. Cells were treated with various doses of lignan SDG (10-50 μM) for 6 hours before irradiation (2, 4, 6, and 8 Gy). The lignan dose was selected based on animal studies to be within the reached physiological level in the blood circulation when 10% flaxseed was ingested. Cells were irradiated at a dose rate of 1.7 Gy / min using a Mark1 cesium (Cs-137) irradiator (JL Shepherd, San Fernando, Calif.). Colonies were stained and counted 10-15 days after irradiation and viability was calculated.

Comet analysis Exponentially proliferating cells were cultured, treated with SDG (50 μM) at different time intervals, and then irradiated (2 Gy). Cells were processed for comet assay according to the manufacturer's instructions (Trevigen, Gaithersburg, MD). Briefly, cells (1 × 10 ⁵ cells / mL in PBS) were mixed with LMAgarose® (1:10, v / v) and immediately pipetted onto CometSlide ™. The cells were then lysed (4 ° C., 30 minutes) and kept in the dark for rewinding (room temperature). Electrophoresis was performed at 18 volts (200 amperes) for 25 minutes in a horizontal electrophoresis unit. Slides were washed twice with DW, fixed with 70% ethanol, and dried at 45 ° C. DNA was stained with SYBR green (Trevigen). At least 150 cells per group were scored. Visual analysis of cells and comet tail length were measured using comet image analysis software (Comet Assy IV, Perceptive Instruments Ltd, Haverhill, UK). Images were captured with an Olympus IX51 fluorescent microscope using a monochrome CCD FireWire camera.

Immunostaining and flow cytometry of γ-H2AX For γ-H2AX immunostaining, cells were seeded on coverslips (5,000 cells / coverslip), pretreated with 50 μM SDG (6 hours), Irradiated (2 Gy). At desired time intervals, cells were fixed (4% paraformaldehyde), washed and blocked with PBST (PBS + 0.1% Triton X-100 with 5% goat serum, 1% BSA). Cells are incubated with γ-H2AX primary antibody (1: 200) at 4 ° C. overnight, followed by washing with PBST (3 × 5 min) and secondary antibody (Alexa fluor®, Invitrogen, CA, USA) And incubated at room temperature for 1 hour. Cell nuclei were counterstained with DAPI and visualized under a fluorescence microscope.

  Cells were trypsinized and washed with PBS for FACS analysis. Cells were then fixed for 45 minutes (Fix / Perm buffer, eBioscience) and then washed with permeabilization wash buffer (BioLegend, USA). Cells are resuspended in 200 μl of rabbit monoclonal phosphohistone γ-H2AX (Ser139) antibody conjugated to Alexa fluor® 488 (1: 100 v / v, Cell Signaling Technology, US) and 30 ° C. at 30 ° C. Incubated for minutes. Cells were washed again with wash buffer and analyzed. Γ-H2AX was measured using CyAn ADP (Advanced Digital Processing) flow cytometer (Dako, Denmark) Coulter, Fullerton, Calif., And positive cells were quantified using Summit Software (Dako, Denmark).

Morphological detection of apoptotic cells Apoptotic cells are characterized morphologically by nuclear and cytoplasmic condensation, membrane blebbing, cell contraction, and degradation of nuclear DNA, initially large fragments. Yes, followed by a fragment of nucleosomes, eventually forming a fully encapsulated apoptotic body. The percentage of cells undergoing apoptosis was determined microscopically from the slide used to detect micronuclei (cytogenetic damage). For each experiment (experiments were performed twice), at least 500 cells were counted and the percent apoptotic cells was determined as follows:
% Cell death = N _a / N _t x100
Where N _a is the number of cells with apoptotic bodies and N _t is the total number of cells analyzed.

Gene expression analysis by quantitative real-time PCR (qPCR) qPCR was performed using TaqMan® Probe-Based Gene Expression Assays provided by Applied Biosystems, Life Technologies (Carlsbad, Calif.). To assess the effect of SDG treatment on apoptotic gene mRNA expression, separate TaqMan® gene expression assays for Bax (Mm00432051_m1) and Bcl-2 (Mm00477631_m1) were performed.

Briefly, cells were pretreated with SDG (50 μM, 6 hours) and irradiated (2 Gy). Total RNA was isolated using RNeasy Plus Mini Kit (Qiagen, Valencia, CA) and quantified using NanoDrop2000 (ThermoFisher Scientific, Waltham, Mass.). Subsequently, reverse transcription from RNA to cDNA was performed on a Veriti® Thermal Cycler using a High Capacity RNA to cDNA kit provided by Applied Biosystems, Life Technologies (Carlsbad, Calif.). qPCR was performed on a StepOnePlus ™ Real-Time PCR System (Applied Biosystems, Life Technologies, Carlsbad, Calif.) using 25 ng of cDNA per reaction well. Gene expression data was normalized to 18S ribosomal RNA, were calibrated against untreated control sample by [Delta] [Delta] _T method.

Apoptosis detection by Western blotting The level of Bax (apoptosis promoter), Bcl-2 (apoptosis inhibitor), cleaved caspase 3, and cleaved poly (adenosine diphosphate ribose) polymerase (PARP) observed using immunoblotting was determined by Apoptosis in lung epithelial cells was determined. Briefly, cells were lysed in PBS containing protease inhibitors. Subsequently, immunoblot analysis of cell lysates was performed using a 10-well SDS 12% NuPAGE gel (Invitrogen, Carlsbad California). Electrophoresis was performed at 200V for 1 hour. Transfer to a PolyScreen PV transfer membrane (PerkinElmer Life Sciences, Boston, Mass.) Was performed at 25 volts for 1 hour. The membrane was blocked overnight in phosphate buffered saline containing 5% nonfat dry milk. Thereafter, the skim milk powder was discarded and the membrane was incubated with the primary antibody. The protein levels of Bax, Bcl-2, cleaved caspase 3, and cleaved PARP were measured using the rabbit anti-mouse monoclonal antibody against Bax and Bcl-2, and the rabbit anti-mouse cleaved caspase 3 (Asp175) monoclonal antibody and the rabbit polyclonal anti-cleaved PARP (214 / 215) Cleavage site-specific antibody was used to detect using dilution recommended by the manufacturer (Cell Signaling Technology, Danvers, Mass.). The membrane was washed 5 times and then incubated for 45 minutes at room temperature in secondary antibody conjugated to horseradish peroxidase. Membrane color was developed using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Boston, Mass.), And the amount of β-actin detected using a specific secondary antibody (Sigma, St. Louis, MO) was detected. Were quantified by densitometric 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).

Statistical data are expressed as mean +/- standard error. Clonogenic assay survival curves were generated using KaleidaGraph software (4.0). Statistical differences between groups were determined using one-way analysis of variance (ANOVA). When statistically significant differences were observed (p <0.05), individual comparisons were made using the Bonferroni / Dunn test (Statview 4.0).

Results SDG treatment enhances the ability of irradiated primary lung cells to colonize Clonogenic survival assays have caused cells to undergo some genotoxic stress after exposure to environmental and pharmaceutical carcinogens, ionizing radiation, etc. Widely used to determine later cell death. In this example, the effect of pretreatment with SDG (10-50 μM) on the radiation-induced reduction of the clonal ability of primary lung cells (epithelial cells, endothelial cells and fibroblasts, respectively) was evaluated. The results show that SDG (10-50 μM) alone did not induce any adverse effects on colony forming ability of all three cell types compared to each untreated control cell (100%). (FIGS. 1A-1C).

  Irradiation treatment significantly reduced the colony-forming ability of epithelial and endothelial cells in a dose-dependent manner (p ≦ 0.01). When cells were treated with SDG before irradiation, the survival rate was significantly improved in all treatment groups (FIGS. 7A and 7B). Maximum protection against radiation-induced loss of clonogenic potential in fibroblasts was observed in the 50 μM SDG pretreated irradiated group (FIG. 7C). We therefore selected this particular concentration of SDG for our subsequent investigation.

SDG prevents the formation of DNA SSB in irradiated primary lung cells. We first consider DNA in all cell types (endothelium, epithelium, fibroblasts) after radiobiologically relevant 2Gy irradiation. A study was conducted to determine the kinetics of damage. As expected, radiation exposure induced significant DNA damage in all cell types compared to their respective unirradiated control cells, as evidenced by an increase in tail moment. The extent of DNA damage was greatest 30 minutes after irradiation. The degree of DNA damage decreased when the time after irradiation reached 60 minutes and then decreased more rapidly by 2 hours after irradiation. We therefore selected a 30 minute time interval for further investigation related to radiation-induced DNA damage (FIG. 8A).

  Radiation exposure (2Gy) resulted in a significant increase in comet tail length compared to unirradiated control cells. Cells were treated at various time intervals (0 hours, 2 hours, 4 hours, 6 hours and 24 hours) prior to irradiation. Pretreatment of cells with SDG (50 μM) significantly suppressed radiation-induced comet tail length in all three types of lung cells at all time intervals. However, maximal protection against radiation-induced DNA tail moment was observed in all cell types during 6 hours of SDG treatment prior to irradiation (FIG. 8B). Representative fluorescence micrographs showing comet tail formation in irradiated (in the presence and / or absence of SDG) lung epithelial cells are shown in the inset of FIG. 8B.

SDG treatment inhibits the formation of γ-H2AX foci in mouse primary lung cells To further verify our hypothesis that SDG can protect against DNA oxidative damage, SDG for induction of γ-H2AX foci The effects of were also evaluated.
The effect of SDG pretreatment on oxidative DNA damage, demonstrated by γ-H2AX formation in mouse primary lung cells after irradiation, was analyzed by standard microscopic image analysis (FIGS. 9, 10) and flow cytometry (FIG. 11). Evaluation was made using both methods. Fluorescence microscopic analysis results show that radiation (2Gy) exposure resulted in a significant increase in the formation of γ-H2AX focus in all three cell types (FIG. 9). The number of focus / cells increased substantially by 15 minutes after irradiation and the peak was 30 minutes (46.7% ± 0.5, 33.3% for epithelium, endothelium and fibroblast, respectively). 6% ± 3.2 and 30.0% ± 1.4 γ-H2AX positive cells), although the number was significantly reduced within 1 hour of exposure, albeit still significantly greater than unirradiated control cells. All values were significantly greater than their respective non-irradiated control cells (p <0.005 for all cell types) (FIG. 9).

  The numbers of γ-H2AX positive cells in irradiated epithelial cells, endothelial cells and fibroblasts were 22.7% ± 2.17, 21.92% ± 2.88 and 22.1% ± 1.9, respectively. As reduced, SDG pretreatment (6 hours before IR) significantly reduced the induction of γ-H2AX (p <0.005 for epithelial cells and p <0.05 for endothelial and fibroblasts). Since the pretreatment of SDG protected all three types of lung cells from radiation-induced DNA strand breaks, the ability of SDG to protect cells from the formation of γ-H2AX foci is independent of cell type It seemed. FIG. 10 shows representative fluorescence micrographs of microscopic analysis of γ-H2AX positive cells in primary lung epithelial cells.

  The protective effect of SDG against inhibition of induction of γ-H2AX positive cells after radiation exposure was further confirmed using flow cytometry. As expected, a similar pattern was observed in the induction of γ-H2AX positive cells after irradiation, but the number of γ-H2AX positive cells was significantly suppressed by SDG pretreatment in all cell types (FIG. 11).

SDG treatment prevents IR-induced apoptotic death of primary lung cells To investigate the cytoprotective effect of SDG from the viewpoint of apoptosis, cell nuclei were stained with DAPI and counted for microscopic visualization. Microscopic analysis showed that control cells had intact chromatin (apoptotic cells, approximately 4-5%), but radiation exposure significantly increased the percentage of apoptotic cells in a time and dose dependent manner (p ≦ 0.05). It showed that. At 24 hours, the percentage of apoptotic cells was observed to be 13.9% ± 1.08 and 15.1% ± 1.95 in epithelial and endothelial cells, respectively. As can be seen in FIGS. 12 (A and B), SDG pretreatment (50 μM) significantly (p ≦ 0.05) blocked the IR-induced increase in the percentage of apoptotic cells (in epithelial and endothelial cells). At 9.9% ± 1.08 and 10.71% ± 1.45 apoptotic cells, respectively). Radiation exposure produced a significant increase of 36.4% and 41.8% of apoptotic cells at 24 and 48 hours, respectively, making it clear that fibroblasts are the most sensitive. Importantly, as shown in epithelial and endothelial cells, pretreatment with SDG significantly reduced the extent of apoptosis even in lung fibroblasts (FIG. 12C).

SDG treatment alters the expression of regulators of apoptosis in mouse primary lung epithelial cells To further elucidate the radioprotective effects of SDG that suppress DNA damage and cell death, we It was hypothesized that the ratio of apoptotic regulator protein could be changed. Therefore, we investigated whether SDG treatment of lung cells in the presence or absence of radiation alters Bax and Bcl-2 gene expression and whether these changes lead to changes at the protein level. Tested. For this, lung epithelium, endothelium and fibroblasts were treated with SDG (50 μM) and enzyme mRNA levels were assessed by qPCR at 6, 24 and 48 hours after IR (FIGS. 13A and B). We found that 2Gy produced an approximately 11-fold increase in proapoptotic Bax mRNA levels at 24 and 48 hours after IR, which was significantly (p <0.05) suppressed by SDG pretreatment. Observed. Alternatively, anti-apoptotic Bcl-2 mRNA levels were not altered by radiation exposure, but rather increased 6.6 and 3.5-fold in SDG pretreated cells at 24 and 48 hours after IR treatment, respectively. . During protein validation by Western blot analysis of changes in Bax and Bcl-2 mRNA levels, we tend to subsequently increase both Bax and Bcl-2 protein levels and decrease their levels in SDG pretreated cells (Figure 13C shows a representative blot, 13D and 13E show quantification of band density). In order to further investigate the underlying mechanisms behind the radioprotection observed by SDG, we determined that the key proteins involved in the apoptotic signaling cascade: the levels of cleaved caspase 3 and cleaved PARP that act as actors The effect of SDG on change was evaluated. Overall, cleaved caspase 3 and cleaved PARP levels increased significantly in irradiated cells at 6, 24, and 48 hours after IR, and their levels tended to decrease in SDG pretreated cells (FIG. 14A shows a representative blot, 14B and 14C show quantification of band density).

Discussion In this example, we showed that FS lignan SDG protects mouse primary lung cells against radiation-induced DNA oxidative damage and apoptotic death. We not only improve SDG pre-treatment as measured by clonogenic survival but also reduce induction of DNA strand breaks (DSB and SSB) and cell death in lung cells Admitted. Importantly, the expression of genes associated with the regulation of apoptosis was also altered by SDG treatment. These findings suggest that SDG may be useful as a non-toxic radioprotectant in radiation scenarios and in improving the therapeutic index of radiation therapy.

  IR-induced cell death is a classic marker of cellular radiosensitivity, which is characterized by a loss of clonogenic survival. Similarly, we also noted a radiation dose dependent decrease in clonogenic potential in all three mouse lung cells. This decrease in clonogenic ability was significantly attenuated by SDG pretreatment (FIG. 7). Cellular DNA is a major target for IR-induced damage in cells. The ROS generated during exposure causes a series of changes in cellular DNA, which ranges from mutations, base damage, cross-linking, SSB and DSB. Damage in the DNA cannot be replaced and therefore needs to be repaired, and if left unrepaired, the cell may induce apoptosis or necrosis. Thus, protection of target cell DNA provides a first-line defense against genotoxic invasion. ROS generation occurs within seconds of exposure and persists for several minutes after irradiation. Thus, initial radiation reactions such as immediate DNA damage occur within minutes after radiation exposure. Similarly, in time-dependent kinetic studies of irradiated cells, we also show that the extent of DNA strand breaks reaches its maximum level within 30 minutes and then decreases, as evidenced by the length of the comet tail This was confirmed (FIG. 8A).

  The comet assay is a sensitive technique for the detection of DNA damage / repair at the cellular level and has been widely used for DNA strand break studies. Polyphenols have the ability to protect normal tissues or cells from the harmful effects of radiation by reducing ROS-mediated oxidative DNA damage. In this study, we also evaluated the role of SDG on IR-induced SSB in mouse primary lung cells by using a standard alkaline comet assay. Our results are consistent with other reports showing similar radiation-induced DNA SSB protective effects on other phenolic components such as chrysin and epicatechin.

  Although SSB is easily repaired by cells, DSB is more difficult for cells to repair and is more likely to mutate, and thus DSB often means a lethal cell event. H2AX molecules are phosphorylated (γ-H2AX) along the megabase long chromatin domain for each DNA double strand break introduced by irradiation, and loss or dephosphorylation of γ-H2AX is temporally related to DNA repair. Correlate. We now report that SDG protected cellular DNA from IR-induced DSB in all three types of tested cells. Our results are consistent with several other studies that also show that polyphenols such as resveratrol and green tea catechin protect cells from IR-induced DNA strand breaks. However, in the presence of radiation ratio, SDG did not show any toxic effect on any of these cells. Since DNA oxidative damage is believed to be a precursor to many cancers, the reduction of such damage by SDG acting as an antioxidant can lead to a reduction in cancer risk.

  It is known that IR-induced apoptosis in cells and tissues is due in part to the induction of DNA damage in the cells. Several mechanisms (O6-methylguanine, base N alkylation, bulky DNA adduct, DNA cross-linking) are involved in DNA damage-dependent apoptosis, but DNA double-strand breaks are major in inducing apoptotic cell death Play an important role. In this study, pretreatment of cells with SDG protected against radiation-induced apoptosis in lung cells and suppressed comet tail length and γ-H2AX focus formation. Thus, the protective effect of SDG against radiation-induced apoptotic cell death may be attributed to its ability to reduce cellular oxidative stress, repair ion homeostasis and prevent DNA damage. These findings are consistent with the anti-apoptotic properties of edible whole-grain flaxseed in the investigation of ischemia-reperfusion and radiation-induced tissue damage in our mice, and SDG mediates a protective anti-apoptotic effect in tissue. It is suggested as a promising bioactive ingredient.

  Since IR reduces the level of antioxidant enzymes in the cellular environment, pretreatment of cells with antioxidants further interferes with ROS, thereby reducing the risk of interaction between ROS and biomolecules. In the previous example, we previously reported that pre-feeding animals with a diet containing flaxseed lignan complex increased the level of protective phase 2 antioxidant enzymes in mouse lungs. A hypothetical mechanism for these actions involves antioxidant response element (ARE) -mediated activation of transcription induction. In addition, Nrf2 also initiates de novo synthesis of various antioxidant enzymes involved in protection against oxidative stress-mediated cytotoxicity. Several other reports also suggest that polyphenols such as curcumin and EGCG exert protective effects against oxidative stress, suggested by their ability to induce phase 2 antioxidant enzymes.

  It is clear from the results that SDG reduces cell death in mouse primary lung cells due to its antioxidant properties and protective effects on DNA. Our findings report that ellagic acid, a type of polyphenol found in pomegranate, protects human keratinocytes from UVA-induced oxidative stress and apoptosis through elevated expression of HO-1 and Nrf-2 genes The results of Hseu et al. (Food Chem Toxicol 2012, 50: 1245). Our investigation provides new evidence that the radioprotective effects of SDG in suppressing IR-induced oxidative damage and apoptotic cell death may be due to direct induction of the antioxidant defense system.

  Taken together, these results indicate that flaxseed lignan SDG in flaxseed has strong radioprotective properties that appear to contribute to the protective effect of whole grain observed in animal studies.

Example 3
Secoisolarisiresinol diglucoside (SDG) removes hypochlorite ions: a novel mechanism of SDG protection of genomic DNA from radiation The powerful oxidant hypochlorous acid (HOCl) is physiological Produced by neutrophils by activated myeloperoxidase, which catalyzes the reaction between chloride ions present in water and hydrogen peroxide (H ₂ O ₂ ). Activated neutrophils _H 2 _{O 2} and superoxide anion _{O 2} · ^- to generate. At physiological pH, there is a mixture of both HOCl and hypochlorite ions (ClO ⁻ ). HOCl kills microorganisms by oxidative damage. However, excessive production is known to cause tissue damage. Hypochlorite modifies adenine nucleotides, resulting in the formation of chloramine, which appears to be the primary mechanism of neutrophil-mediated toxicity.

HOCl and its conjugate base ClO ⁻ have been shown to oxidize amino acids, peptides, and lipids and to chlorinate bases in cellular DNA and RNA. The HOCl / ClO ⁻ reaction modifies both purine and pyrimidine nucleotides at the position of the guanine and thymine endocyclic —NH groups and the NH ₂ group of guanine, adenine and cytosine derivatives, such as (RNHCl) and RR′NCl Chloramine is formed. The primary modified bases were found to be 5-chlorocytosine, 8-chloroadenine and 8-chlorologinine in SKM-1 cell DNA and RNA.

It is known that gamma rays can ionize atoms and molecules. In biological systems or solutions, ionizing radiation generates hydroxyl radicals (.OH), which are believed to be the source of ionizing radiation-induced damage to cellular components including lipids, proteins and DNA. However, these extremely unstable radicals can be removed by Cl ⁻ ions present at very high concentrations in physiological fluids. This produces a reactive chlorine-containing intermediate, in which relatively stable ClO ⁻ is a radiation-derived poison. In chloride-containing solutions, ClO ^- and other oxidative active chlorine derivatives are formed as products of radiolysis. They can contribute to the suppression of the physiological functions of the organism. We therefore propose that radiation-induced DNA or protein damage is mediated in part by radiation-generated ClO ⁻ .

Two diastereomers of chemically synthesized SDG have recently been shown to be equally potent in their antioxidant properties, free radical scavenging properties and DNA protection properties. This study, very using novel fluorescent probes specific, saline by γ ray induced ClO ^- evaluating the SDG respect radiation protection of DNA from generation. Hypochlorite-specific 3 ′-(p-aminophenyl) fluorescein (AFP) and hydroxyl radical sensitive 3 ′-(p-hydroxyphenyl) fluorescein (HPF) are related to the measurement of reactive oxygen species (ROS), Provides greater specificity and reproducibility.

Materials and Methods Chemicals ROS-labeled probes APF and HPF, plasmid DNA (pBR322), 1 kb plus DNA ladder were purchased from Invitrogen (Life Technologies, Carlsbad, Calif.).

Measurement of hypochlorite The fluorescence of the ROS probes AFP and HPF in PBS was measured by excitation / emission at 490 nm / 515 nm in the presence of hypochlorite or after gamma exposure. Data are expressed as relative fluorescence units (RFU).

Gamma-ray-induced formation of hypochlorite by measuring taurine chloramine Taurine chlorination was measured using a TMB assay. Data are expressed as taurine chloramine (absorbance) as well as ClO ⁻ concentration (μM).

Hypochlorite-induced damage to calf thymus and plasmid DNA Calf thymus or plasmid DNA was incubated with hypochlorite at 37 ° C. for 2 hours. DNA samples were subjected to agarose (1%) gel electrophoresis and analyzed.

Measurement of hypochlorite-induced chlorination of 2-aminopurine (2-AP) Fluorescence spectrum with 2-AP in PBS exposed to hypochlorite and maximum emission at 374 nm from 260 to 390 nm Was recorded. 2-% change in AP was calculated.

Statistical analysis of data Data obtained are expressed as mean ± standard deviation. Data were subjected to one-way analysis of variance (ANOVA) with post hoc comparison using Bonferroni's corrections using the Statview Program. A p value ≦ 0.05 was considered significant.

Results In this study, we as a possible mechanism of DNA protection from radiation exposure in a physiological solution, radiation-induced ClO ^- examined the ability of SDG to remove.

SDG removes hypochlorite ions The specificity of selected fluoroprobes was evaluated using sodium hypochlorite. FIG. 15A shows a linear increase in the fluorescence intensity of APF with increasing ClO ⁻ concentration (1-4 μM). Importantly, the increase in HPF fluorescence intensity is negligible, indicating that the fluorescence of APF is primarily ClO ^- dependent. The ClO ⁻ dose was selected to be within this range for subsequent experiments. Next, the SDG (commercially available), ClO ^- to assess the ability of SDG to remove. Indeed, SDG reduced ClO ⁻ in a dose-dependent manner (FIG. 15B). Finally, we evaluated the ClO ^- removal effect of synthetic SDG diastereomers SDG (S, S) and SDG (R, R). In 0.5μM, SDG (R, R) and SDG (S, S), and SDG (commercially available) is, ClO ^- similar to silibinin being established as a removing agent, ClO at equipotent ^- was removed ( FIG. 15C).

SDG, as evidenced by an increase in fluorescence intensity of the APF to remove γ-ray induced hypochlorite, radiation ClO ^- generates the in dose-dependent manner. In particular, 50 Gy gamma-induced APF and HPF (FIG. 16A) were dose-dependently reduced by SDG (FIGS. 16A and B). The first slope of decrease in APF fluorescence (1,816.30) is greater compared to HPF (695.75), indicating a selective removal effect of SDG on ClO ⁻ (inset, FIG. 16A). The effect of SDG on APF was significantly more pronounced compared to HPF (FIG. 16B). SDG reduced ClO ⁻ production from radiation exposure (25 and 50 Gy), indicated by increased APF (FIG. 16C) and HPF (FIG. 16D) fluorescence (p <0.05). The APF / HPF ratio is decreased by SDG, indicating selective removal of ClO ⁻ .

Hypochlorite dose dependent increase radiation induced ClO in the chlorination of taurine ^- for is determined that the chlorinating NH groups in biological molecules, we radiation induced taurine Chlorination was evaluated. The results (FIG. 16E) show that, in saline, γ-rays significantly increased taurine chloramine formation at 50, 100, and 200 Gy, and γ-rays induced ClO ⁻ production, which was dependent on chloride concentration. Shown (FIG. 16F). This result provides strong evidence that radiation induces ClO ⁻ in physiological solutions that can damage biomolecules.

Or induces damage to the genome (Figure 17A～D) and plasmid (Fig 17E~F) DNA ^- SDG is we protect the hypochlorite-induced damage to the calf thymus and plasmid DNA, ClO Clarified. Indeed, ClO ⁻ induced DNA fragmentation in a dose-dependent manner (FIGS. 17A, B) and increased low molecular weight fragments. Damage to genomic DNA exposed to 0.5mM hypochlorite 1.0μM is by all SDG (commercially available or synthesized), known anti quercetin oxidant and ClO ^- up Remover silibinin comparable level Reduced (FIGS. 17C, D). Similarly, damage to plasmid DNA by ClO ⁻ was also reduced by SDG (FIGS. 17E, F). In particular, we evaluated the amount of supercoiled (SC) plasmid DNA compared to damaged open-coil DNA, which has a different migration pattern on agarose gel electrophoresis. The presence of SDG at 25 μM reduces ClO ^- induced damage to plasmid DNA, and the DNA is mostly supercoiled (81.3% ± 9%) compared to OC (18.6% ± 9.4%). 9.4%).

ClO to protective effect DNA of SDG for hypochlorite-induced chlorination of 2-AP ^- for the damage to clarify whether caused by nucleobases modification, we a fluorescent analog of purine The ClO ^- induced chlorination of certain 2-APs was evaluated. Hypochlorite reduces 2-AP fluorescence when given at a concentration (10 μM) equivalent to that produced by radiation exposure (FIGS. 15A, 16A, 17C), which is pretreated with SDG (60 seconds). ) (18A, B). Most importantly, post-treatment with SDG was significantly different from hypochlorite-induced 2-AP modification when SDG was added +15, +30, +60, +120, +180 or +300 seconds after exposure to ClO ⁻ . (Figures 18B and C). These results demonstrate the nucleobase protective properties of SDG against hypochlorite-induced modification of purine bases.

Protective effect of SDG against gamma-induced chlorination of 2-AP To clarify whether radiation induces nucleobase chlorination, we have the same system as above, namely 2-AP fluorescence (FIG. 19A )It was used. Indeed, exposure to gamma radiation resulted in a dose-dependent decrease in fluorescence intensity (similar observations in the presence of ClO ⁻ (FIG. 18A)), which was blocked by SDG (FIGS. 19A, B). These results indicated that gamma radiation induces nucleo-based chlorination, demonstrating the protective properties of SDG against such radiation-induced modifications of purine bases.

Proposed mechanism of action of SDG to prevent or mitigate nucleobase chlorination by radiation With regard to the mechanism of recovery or inhibition of N-chlorinated nucleobase, we have N-Cl-molecules via electron-rich aromatic rings Two-electron reduction or one-electron reduction of N-radicals suggests that it then deprives hydrogen atoms from the —OH group in the phenolic SDG component. Mechanism 1 (FIG. 20) shows the proposed mechanism of SDG protection. The results of our investigation provide evidence for a new mechanism of SDG radioprotection by removing hypochlorite ions and protecting against radiation-induced DNA damage.

Discussion The results of this study provide the following evidence: i) Known lignan antioxidant and free radical scavenger SDG is a hypochlorite ion produced in physiological solutions by chemical means and radiation. Ii) SDG protected genomic and plasmid DNA from hypochlorite-induced damage; iii) Mechanism of SDG protection (protection or recovery from hypochlorite-induced DNA damage) Includes removal of hypochlorite and regeneration of the amino (-NH) group from chloramine (-NCl) on the nucleobase; iv) exposure to gamma radiation resulted in increased taurine chloramine formation; v) Exposure to gamma radiation resulted in increased chlorination of purine bases, which was blocked by SDG; vi) The effects of SDG were applied when applied before or after exposure. Equally effective in protecting DNA from hypochlorite-induced damage, ie SDG can function as a nucleo-based chlorination protector and / or mitigator; vii) commercial SDG, silibinin Compared to quercetin (natural antioxidant flavonoids), synthetic SDG (S, S) and SDG (R, R) diastereomers are capable of removing hypochlorite ions and hypochlorite-induced DNA damage Was equally powerful in preventing. These results demonstrate the protective and mitigating properties of SDG against nucleo-based hypochlorite-induced modifications.

  A mouse model of chest radiation injury was used to determine the tissue radioprotective role of whole grain edible flaxseed, a cereal rich in lignan polyphenols, as well as a flaxseed lignan formulation rich in SDG. These studies have defined the radioprotective and radiation mitigating properties of lignan SDG against in vivo radiation-induced tissue damage. Extraction, purification, or synthetic flaxseed SDG is a powerful antioxidant in vitro as well as in vivo. To investigate the therapeutic potential of SDG, we synthesized SDG by a novel chemical reaction and demonstrated the antioxidant properties of synthetic SDG (R, R) and SDG (S, S), their reducing power, metal chelates. Clarification potential and free radical removal activity against OH, peroxy and DPPH radicals were clarified. We also show the radioprotective properties of synthetic SDG (R, R), SDG (S, S) enantiomers, their potential to prevent gamma radiation-induced damage to plasmid DNA (pBR322) and calf thymus DNA. It was demonstrated by evaluating.

We have also already achieved maximum radioprotection of genomic DNA by SDG at a concentration of about 5.0 μM, which is due to EC ₅₀ values for their free radical scavenging and antioxidant effects, usually in the range of 130-200 μM. It showed a low concentration. It is interesting to note that the maximum efficacy of SDG in removing radiation-induced hypochlorite, as confirmed in this study, falls in the range of 0.5-5 μM. This suggests that the DNA protection effect of SDG is due in part to the removal of harmful ClO ⁻ . The results showing the protective effect of SDG against hypochlorite-induced modification of 2-AP indicate that SDG protects DNA by preventing hypochlorite-induced damage to the nucleobase.

  HOCl is produced by myeloperoxidase of activated neutrophils using hydrogen peroxide produced by NADPH oxidase and chloride ion as a substrate. HOCl can chlorinate and oxidize the nucleobase. Since HOCl can chlorinate the nucleobase, this can cause genotoxicity. Chlorinated nucleosides have been identified and have been implicated in inflammation and cancer.

  There may be several possible mechanisms shown below that allow SDG to protect DNA from radiation-induced damage. 1) Mechanism by removing hypochlorite ions that cause nucleobase chlorination and oxidation; 2) React with chloride ions to form hypochlorite • Remove OH free radicals Mechanism. We have observed that the production of OH is dramatically reduced in the presence of chloride ions in phosphate buffered saline (PBS) as well as in saline alone (preliminary experiment). Given the high concentration of chloride ions in physiological fluids in the body, hypochlorite production and the importance of hypochlorite-induced damage to cellular components, including DNA, are the main mechanisms of radiation damage. I will. 3) Mechanism by binding to DNA base pairs, like some flavonoids such as luteolin, kaempferol and quercetin; 4) on purine and pyrimidine bases, especially at positions C5, C6 and C8 and deoxyribose Mechanism by preventing proton removal or addition of OH. These mechanisms have been proposed for protection from free radical-induced DNA damage. 5) Finally, the mechanism by reduction of the chloroamine formed and thus by regenerating internal and external amino groups in the nucleic acid. Thus, as a hypochlorite ion scavenger and as an antioxidant and free radical scavenger and nucleo-based protectant from hypochlorite-induced chlorination, SDG is a DNA radioprotectant and radiation mitigator Can function as.

Hypochlorite is present in solution in a pH dependent amount as a mixture of hypochlorite anion (ClO ⁻ ), hypochlorous acid (HClO) and free chlorine (Cl ₂ ). At physiological pH, ClO ^- and HClO are major molecule. • Unlike strong one-electron oxidants such as OH, hypochlorite is a two-electron oxidant, and is less reactive and more selective than OH. Hypochlorite can chlorinate electron-rich aromatic rings and NH-compounds. Hypochlorite oxidizes primary and secondary alcohols as well as benzylmethylene and tertiary methine groups and phenol. The first step of the above reaction is chlorination followed by hydrolysis / HCl removal. SDG contains all of the above reactive sites except the amino group, which makes the SDG molecule a powerful hypochlorite scavenger. In Mechanism 1 (FIG. 20), we proposed a novel mechanism of DNA protection by SDG using nucleobase one-electron or two-electron reduction.

In summary, we have shown that SDG removes hypochlorite (ClO ⁻ ) ions and prevents radiation-induced hypochlorite and DNA damage. Since hypochlorite ions are known to modify DNA bases by chlorination / oxidation and subsequently cause DNA damage, our findings indicate that incidental to radiation therapy or radiation for cancer treatment It shows that SDG may be useful as a radioprotectant for normal tissue damage associated with exposure.

Example 4
Flaxseed and its lignan protect against the action of benzoalphapyrene Flaxseed, SDG and lignan derivatives alleviate lung tumor formation due to tobacco and other environmental carcinogens by inhibiting multi-stage carcinogenic processes ( FIG. 21). In this example, experiments are provided that show that lignan SDG has chemoprotective activity in animal models, through modulation of the phase 2 detoxification pathway regulated by Nrf2, and possibly other mechanisms. The protective effects of SDG are mediated by direct ROS ablation and / or indirect antioxidant / anti-inflammatory properties, as well as reduced carcinogen toxicity and DNA damage.

  As detected by redox-sensitive fluorescent dyes (FIG. 23), exposure of mouse epithelial cells to tobacco and the environmental carcinogen benzoalphapyrene (BaP) induces harmful reactive oxygen species (ROS). As early as 2 hours after exposure to carcinogens, a clear increase in fluorescence intensity indicates the production of ROS in the cells.

  To show that SDG reduces DNA oxidative damage induced by carcinogens in cells, SDG (10 μM) is added to human epithelial cells (A549) exposed to 25 μM BaP and mass spectrometry is used. Oxidative damage to DNA was detected as indicated by the presence of 8-oxo-7,8-dihydroguanine (8-oxo-dGuo). SDG reduced DNA damage at 3 and 6 hours after carcinogen exposure (FIG. 22). Similarly, to show that SDG blocks ROS production from carcinogen exposure, mouse epithelial cells were treated with 10 or 20 μM BaP and increasing concentrations of SDG (0, 0.1, 0.5, 1, (5 μM SDG) and ROS was detected after 2 hours. SDG removed harmful ROS to a negligible level (Figure 24).

  Similarly, SDG prevents genotoxic stress in human epithelial cells exposed to BaP. Exposure of cells to potent carcinogens such as BaP induces genotoxic stress indicated by increased levels of p53 protein (FIG. 25). This is mitigated in a dose-dependent manner by the presence of SDG at concentrations of 5, 10, 25 and 50 μM. SDG also prevents oxidative DNA damage in human epithelial cells exposed to BaP. Exposure of cells to potent carcinogens such as BaP induces DNA oxidative damage indicated by increased levels of γ-H2AX, a marker of double-stranded DNA breaks (FIG. 26). This is mitigated in a dose-dependent manner by the presence of SDG at concentrations of 5, 10, 25, 50 and 100 μM. In addition, SDG prevents DNA adduct formation in human epithelial cells exposed to BaP. Exposure of cells to potent carcinogens such as BaP induces DNA adduct formation (FIG. 27). DNA adducts are fragments of DNA that are covalently bound to carcinogens and are directly associated with the development of malignant lesions. DNA adduct levels are reduced by the presence of SDG or its metabolites ED and EL added alone or in combination.

  Flaxseed and its lignan provide protection in a mouse model of chemically carcinogen-induced lung tumors. To mice (A / J strain), tobacco and the environmental carcinogen BaP were intraperitoneally injected at a dose of 1 mg / Kg four times (once a week) (FIG. 28). Mice are started on flaxseed or lignan diet upon exposure. Mice were evaluated at various times after exposure to identify tumor burden, mouse weight, and overall health profile.

Flaxseed reduces tumor burden in mice FIG. 29 shows representative clinical images of mouse lungs months after BaP exposure and edible flaxseed administration. FIG. 30 shows representative H & E stained lung sections of mouse lungs months after BaP exposure and edible flaxseed administration. The nodule indicated by the arrow in mice fed the control diet (upper panel) or flaxseed (lower panel) appears small in mice fed flaxseed. Each panel represents a different animal.

  Histological mouse lung sections were morphologically evaluated for overall tumor area and nodule size using image analysis software (FIGS. 31A and B). There was a significant reduction in the area of the lungs occupied by tumors in mice fed flaxseed diet (p <0.03). Similarly, there was a trend for smaller tumor nodule sizes. Histological mouse lung sections were similarly evaluated morphologically for the total number of tumor nodules per lung (FIG. 32A) and% tumor infiltrating the lung (FIG. 32B). There was a trend for fewer tumor nodules per lung (A) and less tumor infiltration (B) with flaxseed supplementation.

  Finally, it was shown that flaxseed supplementation prevents the wasting effect of lung cancer induced by BaP. Animal weights were measured over a period of 200 days after BaP exposure. Mice fed flaxseed diet and exposed to BaP showed higher weight than mice fed control diet and exposed to BaP (FIG. 33).

Example 5
Flaxseed and its lignans protect cells and tissues from asbestos-induced damage Introduction Malignant mesothelioma (MM) is a destructive, painful and lethal type of cancer that has no real promise of treatment and treatment. is there. The occurrence of MM has been directly linked to exposure to asbestos fibers. Recent research has shown that the development of asbestos-induced cancer is due to chronic inflammation and oxidative tissue damage caused by residual asbestos fibers. Whole grain flaxseed (FS) has known antioxidant, anti-inflammatory and cancer chemoprotective properties. In this example, the ability of FS and its lignan component (FLC) enriched in lignansecoisolariciresinol diglucoside (SDG) added to the diet to prevent acute asbestos-induced inflammation and inflammatory cytokine release is Nf2. ^{+ /} Mut, Cdkn2a ^{+ / mut} mice were tested.

Materials and Methods Mice Diet and Exposure to Asbestos Rodent chow was fed with FS and its lignan component (FLC) enriched in lignansecoisolariciresinol diglucoside (SDG). Mice (Nf2 ^{+ / mut} , Cdkn2a ^{+ / mut} ) were given a 10% FS or 10% FLC supplemented diet one day (+1) or one day before (-1) a single 400 mg asbestos asbestos ip bolus. After 3 days, abdominal inflammation and pro-inflammatory cytokine release were evaluated (see FIG. 34). The NF2 mouse strain was selected because it develops rapidly progressive MM upon exposure to asbestos.

Tissue collection and analysis Systemic levels of flaxseed lignan metabolites such as mammalian lignan enterolactone (EL) and enterodiol (ED) using liquid chromatography, tandem mass spectrometry (LC / MS / MS) (ie, plasma) Was evaluated to confirm that FS is effectively metabolized by the intestinal flora of this mouse strain and that the levels are comparable to those of other mouse models.

  Abdominal lavage was performed using PBS and cytospin analysis was used to determine macrophage (MF) and neutrophil (PMN) levels.

Discussion Using liquid chromatography, tandem mass spectrometry (LC / MS / MS) to assess systemic levels (ie plasma) of flaxseed lignan metabolites such as mammalian lignan enterolactone (EL) and enterodiol (ED). It was confirmed that FS was effectively metabolized by the intestinal flora of this mouse strain and that the levels were comparable to those of other mouse models (FIG. 37). Abdominal lavage levels of macrophages (MF) and neutrophils (PMN) showed that both FS and FLC reduced acute abdominal inflammation induced by asbestos (FIGS. 36 and 38). Furthermore, the levels of pro-inflammatory cytokines TNF-α and IL-1β were also reduced by edible drugs (FIGS. 37 and 38).

  Conclusion: These findings show that the chemoprotective properties of flaxseed and its lignan component extend to protection from asbestos-induced tissue and cell damage. Therefore, flaxseed can be used as an edible drug in chemoprotection of malignant mesothelioma.

Example 6
Optimization of dose and kinetics of SDG administration in vivo This example provides data on pharmacokinetics, bioavailability, and dose response in mice that were orally gavaged with SDG in a water-soluble form .

  Dosing study: SDG doses of 5, 25, 50 and 100 mg / kg body weight were orally administered to mice (each dose n = 5) and 4 hours later, tissues were collected for analysis. Plasma concentrations of SDG and its bioactive metabolites ED, EL and SECO were analyzed by liquid chromatography and mass spectrometry and expressed in ng / mL (FIG. 39). In addition, the gene expression level of Nrf2-regulated antioxidant enzyme (AOE), an intracellular target of SDG and its metabolites, was measured in lung tissue from the same animal using qRT-PCR (FIG. 40). Gene expression levels of HO1, NQO1 and GST increased 2-3 times baseline with only 5 mg SDG / Kg and averaged 6 times baseline with 100 mg / kg SDG. Gene levels are supported by a low but significant increase in protein levels in lung tissue.

  Pharmacokinetic study: After selecting 100 mg / kg, a dose that induces significant AOE expression and at the same time capable of detecting SDG in the circulation, a pharmacokinetic study is performed to target the target tissue (ie lung) The bioavailability and biological effects of were determined. Blood samples and tissues (lung, brain, liver, kidney, spleen) were collected at 15 minutes, 30 minutes, and 1, 2, 4, 6, 8, 12 hours. SDG levels in plasma and lung were determined as described above (Figure 41). A single dose of 100 mg SDG / kg is well absorbed, intact SDG can be detected in the circulation up to 6 hours after administration (FIG. 41A) and detected in flushed, blood-free lung tissue for 4 hours It was possible (FIG. 41B). SDG plasma and lung tissue concentrations reached levels of 0.8 and 12.6 μM in 30 minutes, respectively. Of note, distinct induction of representative AOE gene expression levels (HO-1, NQO1, GST) was significantly elevated (p <0.05) relative to baseline (FIG. 41C). The observed increase in gene expression correlated with the increase in protein levels measured by Western blotting (not shown). Importantly, SDG administered twice daily for 7 days at the same dose showed neither intolerance nor toxicity. These data indicate that this soluble SDG form can provide significant bioavailability and efficacy. Thus, the use of SDG in human therapy is greatly facilitated and is unlikely to be a risk of toxicity. Thus, a dose of 50-100 mg / Kg (1 or 2 mg SDG per day) is sufficient to induce the expected protective effect in the target tissue and can be used for further investigation.

Example 7
Effect of SDG on initiation or promotion of carcinogenesis in BaP-induced lung tumor formation in Nrf2-deficient mice Data from the lung injury model show that dietary FS and FS-derived SDG are pulmonary Nrf2 and Nrf2 activating genes As well as increased expression of the protein. In this example, Nrf2 knockout mice are used to test the hypothesis that activation of this pathway is an important mechanism of SDG chemoprotection, and SDG is active in both the onset and promotion stages of carcinogenesis. Make it clear whether you have it.

The effects of SDG in Nrf2-deficient mice In these experiments, the effects of synthetic SDG administered orally in a BaP-induced lung cancer model in wild type A / J mice were compared to the A / J background (currently maintained at our animal facility). To the Nrf2-deficient mice that were backcrossed. Briefly, one wild type (WT) group of mice and one knockout (KO) group of mice are fed a control diet. One WT group of mice and one group of KO mice are administered SDG via their drinking water to achieve daily SDG consumption of 50 and 100 mg / kg as shown in FIG. 42A (dose in inhibiting lung cancer development). To test the reaction relationship). According to SDG dosing studies and kinetics, SDG should be administered for at least several days (since this was determined to be the minimum time required for the diet to reach steady state in the tissue) and continued 4 times weekly, 1 mg / mouse benzo [a] pyrene (BaP) i. p. It was determined that an injection was necessary. Both 50 and 100 mg / kg SDG administered orally (in oral gavage or in drinking water) can induce phase enzyme expression in the lung (see FIG. 41). Mice were sacrificed at 4, 6 and 9 months (see FIG. 42B), lung tissue a) Tissue assessment and quantification of tumor burden by image analysis, b) Western blot detection of Nrf2-regulated AOE expression and oxidative stress, c) Mice It was collected for 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) levels in tissue and urine, and d) DNA adduct formation. FACS of inflammatory cells such as neutrophils, activated macrophages and T cells using a subset of mice a) tubal alveolar lavage test and b) antibodies against CD11b / Ly6G, CD11b / F4 / 80 and anti-CD3, respectively Analyzes are used to assess lung inflammation.

  Statistics: The number of mice required per group to obtain statistical significance was suggested to be 20 mice / group.

  Analysis: As noted above (FIGS. 29 and 31), WT mice given 50 and 100 mg / kg SDG are expected to reduce tumor number and size, but the results at 100 mg / Kg dose are even more pronounced (Less tumor). In Nrf2 KO mice given a control (no drug), a greater number of tumors are predicted when compared to control fed WT mice due to loss of ability to detoxify BaP. However, since the primary effect of SDG is mediated through Nrf2 activation, tumor reduction is not expected in Nrf2 KO mice receiving SDG (compared to KO mice not receiving the drug). Whatever the decrease observed in tumor burden in Nrf2 / SDG mice is due to other mechanisms that contribute as well, such as the direct free radical scavenging effect of SDG, which are oxidative stress Should be detectable by measurement of other markers.

Role of SDG and Nrf2 activation in the initiation and / or promotion phase of carcinogenesis The above data is expected to provide clear mechanistic data on SDG administration during both the initiation and promotion phase of BaP. It is also interesting to clarify the efficacy of SDG supplementation only during the onset phase (FIG. 42A, plan B) or only during the promotion phase (FIG. 42A, plan C). Therefore, experiments outlined in this schema were performed in wild type A / J mice using these different feeding schedules.

  Analysis: Reduction of tumor number and size is expected when SDG is administered during the promotion or initiation phase. As described above, the study is repeated in Nrf2 KO mice to clarify the contribution of increased phase 2 enzyme expression to this activity. It has recently been reported that Nrf2 has two roles during carcinogenesis, one of which is preventive during the onset of tumor formation and the second promotes malignant progression. These findings suggest the use of Nrf2 to prevent malignant progression in lung cancer while suggesting that Nrf2 activators are better suited for lung cancer prevention. SDG is expected to be effective as a chemoprotective agent. Despite the role of Nrf2 in the anti-carcinogenic effects of SDG, other mechanisms such as, for example, modulation of miRNA in systemic body fluid (blood) or lung tissue may also be involved or more important. .

Effects of SDG adjuvant administration to smokers on systemic and lung-specific biomarkers of oxidative stress Genetic and biochemical clinical trials of oral administration of SDG with genetic and biochemical endpoints are designed .

  Selected group: Group 1 consists of healthy volunteers with no smoking experience. This group allows for the assessment of the true baseline level (probably low) of the gene of interest and the oxidation biomarker to assess the changes induced by SDG supplementation in a “non-induced” environment. The second group of subjects will consist of current smokers. This group is selected for the following reasons: i) they are at high risk of active oncogenesis and are potential subjects in chemoprotection studies, and ii) smoking is its own genomic alteration in the respiratory epithelium As well as inducing active oxidative stress. This group allows profiling of the smoker's genome and oxidative stress markers, and makes it possible to determine what genomic changes are induced by SDG in an already “activated” environment. This group also allows the measurement of SDG reduction of elevated oxidative stress markers in this high risk population. A crossover design is chosen in which each patient also takes both a placebo control and an SDG supplement (both administered in gelatin capsules) (see FIG. 43).

  Dosage: A commercial preparation (BREVAIL ™) was selected to avoid large variability in SDG content and bioavailability was observed in different batches of untreated or ground flaxseed. Alternatively, Misra et al. , Bioorganic & Medicinal Chemistry Letters 2013, (19): 5325 are used. In pharmacological studies, daily dosing with this formulation containing 50 mg SDG produced ENL levels (median, 63 nmol / L) similar to those seen in the highest quintile associated with cancer reduction It has been shown.

  Clinical trial: The trial will be a single center, randomized, double-blind, two-phase crossover trial. There are two three-week treatment periods with a one-month drug holiday (Figure 43). The principle of crossover design is that the comparison of treatments within a subject removes all “subject effects” from the comparison, increasing the efficiency and power of the test. Twenty healthy volunteers (10 smokers and 10 lifetime non-smokers) were randomized and in turn received 50 mg SDG (1 capsule of BREVAIL ™ or synthetic SDG) daily for 3 weeks Followed by a placebo (same capsule without SDG by the same provider (Lignan Research, San Diego, Calif.)) For another 3 weeks after a 1-month withdrawal period, or placebo for 3 weeks, Ingestion of 50 mg SDG for 3 weeks, followed by a monthly drug holiday. Oral epithelium (oral swab), breath condensate, urine and blood are obtained at baseline and at 2, 3, 7, 9, and 10 weeks. Current smoking status is confirmed by the urine cotinine assay. The study is approved by the Pennsylvania State University Institutional Review Board and all participants submit written informed consent. A detailed questionnaire survey will be conducted at baseline, including a comprehensive smoking history and passive smoking (ETS). Participants are examined weekly for investigation of side effects, adherence to study drug intake, data collection on temporary tobacco and drug use, and sample collection.

  Rationale for Statistics / Power Analysis: The primary comparison of interest is the change in urinary oxidative stress as measured by urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo) Changes in breath condensate (experimental) and changes in gene expression levels in the oral epithelium of test subjects after SDG treatment. Twenty patients are assumed to provide reasonable power for this preliminary study. Because of the nature of this test, no correction is applied to multiple comparisons. The test is designed to give an assessment of the effectiveness of the SDG adjuvant. It should also be noted that the power to carry over effects testing is limited, but a 4-week washout period should eliminate these concerns.

Assessment of gene expression changes in oral swab epithelial cells after oral SDG ingestion In these experiments, oral mucosal epithelial cells are used as a substitute for bronchial epithelial tissue. Such cells are more easily obtained from bronchial epithelial cells that require invasive bronchoscopy (ie, simply by wiping with a cheek swab). The validity of this approach in humans is supported by evidence that the oral epithelium can function as a substitute for airway bronchial tissue in gene array studies.

  This approach was further validated by using fed FS in mice. After mice were fed a control or 10% FS diet for 3 weeks, a series of tissues were collected and the expression of two representative Nrf-2 inducible proteins (HO-1 and NQO1) was analyzed by immunoblotting. . There was a significant increase in lung and liver of these markers (data not shown). Importantly, there is a clearly detectable increase in both proteins in the nasal epithelium (FIG. 44), thus demonstrating that nasopharyngeal tissue can also be used as a lung substitute. There is also some workable data that supports this approach in humans. Whole grain FS (40 g daily) was administered to two healthy volunteers. Oral swabs were collected, RNA was successfully extracted, and cDNA was prepared. The relative expression level of the Nrf2-dependent gene HO-1 was measured. As shown in FIG. 45, HO-1 mRNA expression increased 34% in one subject and 63% in the second subject (p <0.05). These data indicate that this approach is feasible and that FS and SDG amplify Nrf2-dependent genes in healthy volunteers.

  In this study, oral swabs were collected at baseline and 2 and 4 weeks after fiber control and FS diet in both control and smoking subjects. Representative antioxidants and phase 2 drug metabolizing enzymes, including mRNA and cDNA generated as described above and subjected to RT-PCR to include NQO-1, HO-1, and glutathione-S-transferase (GST) Assess the relative expression level of. In addition to being “typical” representatives of Nrf2 inducible enzymes, the selected enzymes may also play an important mechanistic role. NQO1 plays a dual role in the detoxification and activation of procarcinogens present in tobacco smoke. The mutant genotype of NQO1 was significantly associated with reduced lung cancer risk in Japanese subjects. HO-1 has also been suggested in genetic studies as a cytoprotectant against oxidants and aromatic hydrocarbons in cigarette smoke. Thus, HO-1 is also an important biomarker for monitoring the effect of flaxseed. Many studies have shown a relationship between GST polymorphisms, smoking, and lung cancer. Importantly, benzo [a] pyrene-derived DNA adducts in lung cells are regulated by detoxification of reactive intermediates resulting from metabolism mediated by both cytochrome P-450 and aldo keto reductase by lung-specific GST. Is Rukoto. Since benzo [a] pyrene is an important lung carcinogen and is present in tobacco smoke, GST is monitored as a biomarker of flaxseed effect in smokers.

  Data analysis: A large pool of cDNA from oral swabs from five healthy non-smoking volunteers is created and used as a “baseline” comparator for analysis. cDNA from all test samples is normalized to this pool using β-actin and GAPDH. After normalization, each test sample is compared to a baseline comparator and the percent or fold change is calculated. The primary analysis is to compare changes in gene expression 3 weeks before and 3 weeks after SDG intake in smokers who have never smoked and current smokers (using paired t-tests). Values are also compared to those of placebo control samples. Additional analysis compares changes in expression by SDG across all time points, including withdrawal values (repeated measures ANOVA) and baseline time points and comparisons between nonsmokers and smokers after SDG and placebo feeding Is done. The response kinetics are established by comparing the 2 vs. 3 week data and the 3 week vs. withdrawal data.

Measurement of oxidative stress reduction by SDG in the same patient population Although it is difficult to measure oxidative stress in human subjects, one of the most established techniques is to measure urinary levels of IsoP. Reliable and reproducible assays have been developed for this marker. Data were obtained from patients awaiting lung transplantation who received 40 g FS for up to 13 weeks. Urinary IsoP was found to decline gradually, but FS feeding showed signs of reduced systemic oxidative stress (some IsoP subcategories were reduced to 26% of pre-FS levels). Representative patient data is shown in FIG. 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. 8-Oxo-dGuo can be detected in biological specimens (urine, liver, lung) using LC-MRM / MS. Data from two human volunteers indicate that daily consumption of 40 g FS for 3 weeks elicits a trend for a decrease in 8-oxo-dGuo, a trend also in lung transplant patients (n = 5) (Fig. 47).

  Test: In the test, urine samples are taken at multiple time points in both control and smoking subjects. Urinary levels of IsoP and 8-oxo-dGuo are measured in a blinded fashion.

Breath condensate to assess pulmonary oxidative stress In view of the inability of these subjects to perform invasive tests such as bronchoscopy, breath condensate (EBC), a non-invasive sample collection technique Are used to investigate lung oxidative stress. Many substances are found in exhaled breath, which can be detected in the liquid obtained by cooling the exhaled breath (ie by condensing). The advantage of this method is that it is non-invasive and simple as a non-invasive sampling method for real-time analysis and evaluation of oxidative stress biomarkers in the lung. Biomarkers of oxidative stress include H ₂ O ₂ , isoprostane (IsoP), malondialdehyde, 4-hydroxy-2-nonenal, antioxidants, glutathione and nitrosated stress markers. Isoprostane levels in EBC are evaluated as a useful non-invasive approach to investigate the effect of FS on lung oxidative stress. 8-IsoP was a measure derived from EBC collected from control subjects and smokers. Levels in non-smokers are uniformly low, approaching test sensitivity (1.2 pg / ml ± 0.6, n = 8), suggesting that EBC is not useful in non-smokers. Levels were measured only in 4 smokers, but interestingly, 2 of these subjects were at very high levels: 16 pg / ml and 6.5 pg / ml. Thus, in these smokers with high baseline levels, EBC is used to track lung-specific oxidative stress.

  Data analysis: Baseline values are measured for each subject and the changes observed at the end of the treatment (3 weeks) are evaluated using a paired t-test. Additional analysis compares changes in expression by SDG across all time points, including withdrawal values (repeated measures ANOVA), and comparisons between baseline and non-smokers and smokers after SDG feeding . We can compare the 2 vs. 3 week data and the 3 week vs. withdrawal data to establish kinetics of response.

Flaxseed Lignans, Chemoprotective Properties of SDG These experiments test the hypothesis that SDG can reduce toxicity from xenobiotics and detoxify carcinogens such as BaP, which is similar to sulforaphane. First, the effect of SDG in vitro on bronchial epithelial cells is evaluated. Cytotoxicity tests such as the MTT assay are performed for all SDG doses described below. Second, SDG administered orally in a BaP mouse model is evaluated.

Evaluation of SDG-mediated detoxification of BaP and induction of Nrf2-mediated phase II enzyme expression in normal mouse and human primary lung epithelial cells When BaP (10 or 20 μM) is metabolically activated by epithelial cells Induces ROS in a dose- and time-dependent manner (FIG. 23). In mouse studies, an immortalized C10 mouse bronchial cell line (derived from Balb / C mice) is used. The reason is that these cells are non-tumorigenic, are contact-inhibited and contain all Nrf2 regulatory mechanisms under investigation. A549 cells (transformed but highly differentiated lung cancer cell line used in human type 1 alveolar epithelial cell models (see data in FIGS. 22-23) and, importantly, primary Similar investigations are performed using bronchial epithelial cells).

To assess direct ROS interference, a series of clinically significant concentrations (0.1-10 μM) of SDG were added simultaneously with 10 μM B [a] P, showing direct antioxidant activity. Measured for decrease in H ₂ DCF fluorescence. Liquid chromatography / multiple reaction monitoring mass spectrometry (LC / MRM-MS) is also for measuring the GSSG: GSH ratio. BaP can also rapidly induce DNA adduct formation, which can be measured using LC-MRM / MS (Figure 22). BaP-induced DNA oxidative damage caused by BaP can be induced by 8-oxo-dGuo formation, tail moment of individual cells using standard comet image analysis (data not shown), or immunoblots for detection of γ-H2AX. Concentration analysis (data not shown). To assess DNA adducts and oxidative changes, a series of concentrations of SDG are added simultaneously with 10 μM B [a] P and these parameters are measured. In addition, MTT cytotoxicity assays are performed to assess direct SDG effects on these cells in the absence and presence of carcinogens (only FS and FLC can be tested in vivo). Next, the ability of purified SDG to upregulate phase II enzymes in bronchial epithelial cells is tested. In particular, the induction of GST-Ya and NQO-1 message and protein after 0, 1, 2, 4, 24, 48 and 72 hours of incubation with lignan SDG (0.1-10 μM) treatment was measured. The These enzymes are selected as characteristic ARE-regulating enzymes characteristic of the phase 2 system. RT-PCR and immunoblot analysis are performed. After identifying the time course of increased phase 2 enzyme expression, the epithelial cells are pretreated with SDG and BaP is added at the time of maximum phase 2 induction. The assay is used to assess oxidative stress, DNA damage, and cell death.

  Data analysis, predicted results and additional studies: The first set of studies defines the direct antioxidant effect of SDG. A second set of studies is performed at a later point in time when the SDG itself disappears (thus there is no direct antioxidant effect), clarifying the effect of SDG due to its second phase enzyme-induced effect. Additional measurements of oxidative stress and inflammation were made to measure plasma oxidative stress (plasma malondialdehyde) and pro-inflammatory stress markers (eg, IL-6, IL-1α, IL1β, TNF-α, C reactivity) Protein) due to SDG.

Chemoprotective properties of SDG in a mouse model of A / J mouse chemical carcinogen-induced lung tumor formation Progression of BaP-induced carcinogenesis in A / J mouse lungs with the same study design as above (FIG. 42A, Plan A) The effect of orally administered SDG on SDG supplemented mice are directly compared to controls. Follow-up studies were conducted, including studies using SDG only during the start phase (FIG. 42A, Plan B) and tests using SDG only during the accelerated phase (FIG. 22A, Plan C).

Statistics: Animal studies typically include 3 or more groups of 20 mice (eg, control versus 50 mg / Kg SDG) and are analyzed using ANOVA or other suitable linear model. All calculations assume a two-sided test, an alpha level of 0.05, and a power of at least 80%.

Example 8
Effect of SDG on asbestos-induced carcinogenesis SDG or flaxseed diet reduces asbestos-induced ROS / inflammation, 1) ROS, 2) reduced cytokines, 3) reduced HMGB1, 4) less oncogenic focus, and 5) It is assumed to result in fewer tumors. The underlying hypothesis is that reduced inflammation and oxidative stress will result in reduced malignant transformation of cells and less tumor burden (see FIG. 48).

To test the hypothesis that flaxseed lignan SDG would reduce asbestos-induced inflammation and oxidative and nitrosative stress, murine peritoneal macrophages were treated with various concentrations of asbestos asbestos fibers (10, 20, 30, and 40 ng / cm ² ). At various times after exposure to asbestos (3, 4, 6, and 8 hours), cells were incubated with SDG (50 μM) and supernatants were collected after 24 hours for inflammatory cytokine secretion and nitrosation / oxidative stress. It was detected (FIG. 49).

SDG reduces asbestos-induced ROS secretion by human mesothelial cells in vitro (FIG. 50). Experimental plan for detection of asbestos-induced ROS in cells: HM cells (human mesothelial) or mouse macrophages were plated in 20% DMEM for 24 hours; 20 μM DCF in HBSS was added for 1 hour; Supernatants were replaced with 1% DMEM containing ₂ O ₂ (100 μM), asbestos (10 μg blue asbestos / cm ² ) and / or SDG (50 μM); and fluorescence levels were monitored up to 36 hours after the start of asbestos exposure. The results show that asbestos and H ₂ O ₂ induce high levels of ROS, but the addition of SDG into the medium significantly reduced ROS levels to baseline levels (FIG. 50).

Asbestos-induced oxidative stress (ROS release) in cultured untreated macrophages was evaluated. Cells were treated with the ROS sensitive dye H2DCFDA for 30 minutes, then exposed to vehicle, 40 μg / cm ² asbestos fiber, or 4 uM hypochlorite solution, and fluorescence intensity measured spectrophotometrically over 90, 150 minutes and 27 hours did. Asbestos-induced ROS was generated immediately after asbestos exposure and persisted during the observation period (FIG. 51).

SDG administered to macrophages several hours after exposure to asbestos reduces oxidative stress (FIG. 52). Female C57 / B16 mice were injected with 2 mL of thioglycolate and peritoneal macrophages were harvested 3 days later. 2 million cells per well were seeded in 6-well plates and exposed to 20 μg / cm ² asbestos. Cells were treated with 25 or 50 μM SDG (synthetic SDG) at 3, 4, 6, or 8 hours after asbestos exposure. Cells were harvested 24 hours after asbestos exposure. Analysis was performed on frozen supernatant samples. The results show that malondialdehyde, a marker of lipid peroxidation, increased with asbestos exposure time (FIG. 52A). 25 and 50 μM SDG added to the cells significantly reduced the level of MDA (p <0.05) (FIG. 52B).

SDG administered to macrophages hours after exposure to asbestos reduces nitrosation stress (FIG. 53). To assess SDG in reducing nitrosation stress in cells following asbestos exposure, female C57 / B16 mice were injected with 2 mL thioglycolate and peritoneal macrophages were harvested 3 days later. 2 million cells per well were seeded in 6-well plates and exposed to 20 μg / cm ² asbestos. Cells were treated with 25 or 50 μM SDG (synthetic SDG) at 3, 4, 6, or 8 hours after asbestos exposure. Cells were harvested 24 hours after asbestos exposure. Analysis was performed on frozen supernatant samples. The results show that nitrite, a marker of nitrosation stress, increased with asbestos exposure time (FIG. 53A). 25 and 50 μM SDG added to the cells significantly reduced the level of MDA (p <0.05) (FIG. 53B).

SDG administered to macrophages hours after exposure to asbestos reduces inflammatory cytokine (IL-1β) secretion (FIG. 54). To assess SDG in reducing inflammatory cytokine (IL-1β) secretion in cells following asbestos exposure, female C57 / B16 mice were injected with 2 mL thioglycolate and peritoneal macrophages harvested 3 days later did. 2 million cells per well were seeded in 6-well plates and exposed to 20 μg / cm ² asbestos. Cells were treated with 25 or 50 μM SDG (synthetic SDG) at 3, 4, 6, or 8 hours after asbestos exposure. Cells were harvested 24 hours after asbestos exposure. Analysis was performed on fresh supernatant samples. The results show that IL-1β, a pro-inflammatory cytokine marker, increased with asbestos exposure time (FIG. 54A). 25 and 50 μM SDG added to the cells significantly reduced IL1β levels (p <0.05) (FIG. 54B).

SDG administered to macrophages hours after exposure to asbestos reduces inflammatory cytokine (TNF-α) secretion (FIG. 55). To assess SDG in reducing the secretion of inflammatory cytokines (TNF-α) in cells following asbestos exposure, female C57 / B16 mice were injected with 2 mL thioglycolate and peritoneal macrophages were harvested 3 days later. did. 2 million cells per well were seeded in 6-well plates and exposed to 20 μg / cm ² asbestos. Cells were treated with 25 or 50 μM SDG (synthetic SDG) at 3, 4, 6, or 8 hours after asbestos exposure. Cells were harvested 24 hours after asbestos exposure. Analysis was performed on fresh supernatant samples. The results show that TNF-α, a marker for pro-inflammatory cytokines, increased with asbestos exposure time (FIG. 55A). 25 and 50 μM SDG added to the cells significantly reduced the level of TNF-α (p <0.05) (FIG. 55B).

  Briefly, the in vitro experiments of this example show that (1) SDG blocks asbestos-induced ROS in human mesothelial cells and mouse untreated macrophages; (2) SDG is exposed to asbestos in the mouse peritoneum Inhibits inflammatory cytokine secretion by macrophages; and (3) shows that SDG blocks oxidative stress (lipid peroxidation) and nitrosation stress (nitrite levels) in mouse peritoneal macrophages exposed to asbestos . These in vitro experiments support in vivo experiments that demonstrate the usefulness of SDG in reducing chronic inflammation and ultimate malignancy from asbestos exposure.

  Therefore, SDG is tested in asbestos-induced mesothelioma using two mouse models, where the mice are genetically predisposed to develop mesothelioma after asbestos exposure. These models are used to assess the acute effects of flaxseed and SDG on a single dose of asbestos in mice. We also test whether flaxseed and SDG inhibit tumor development in these models of asbestos-induced rapid progression MM (FIG. 56).

  Flaxseed lignan diet (FLC) rich in SDG reduces asbestos-induced abdominal inflammation in MEXTAG mice (FIG. 57). Briefly, six male MEXTAG mice (10-12 weeks of age) were injected ip with 400 μg of asbestos asbestos in a volume of 0.5 mL. Prior to asbestos injection, half of the mice received an SDG-rich FLC diet (35% SDG) for 2 weeks, and the remaining mice received a standard diet. Three days later, the mice were washed with 5 mL PBS and the washings were collected for supernatant and cell count. Cytospin was performed, 3-5 separate fields were counted, and the% difference of total cells was calculated. Compared to 0% FS-fed mice, 10% FLC-fed mice had a 26% decrease in WBC in abdominal lavage fluid (p = 0.014) (FIG. 57).

  An acute phase study was conducted in 7-week-old male NF2 (129sv) (+/−) mice exposed to asbestos to evaluate the effects of flaxseed and lignan SDG formulations administered by diet. The experimental design of NF2 mouse asbestos exposure and flaxseed / SDG lignan blend evaluation is shown in FIG. Briefly, male NF2 (129SV) (+/−) was exposed to 400 μg asbestos via intraperitoneal injection on day 0. Mice started the test diet (0% FS, 10% FS, 10% FLC; n = 2 mice in each group) 24 hours before asbestos exposure (day -1) and were sacrificed 3 days after asbestos exposure . Abdominal lavage (AL) was performed using 5 mL 1 × PBS. The influx of inflammatory cells peaked by 3 days after asbestos exposure and gradually decreased by 9 days. Therefore, in all subsequent experiments, 3 days was chosen as the time point for assessing inflammation (FIG. 59).

  Flaxseed and its SDG-rich lignan component reduced asbestos-induced inflammation (younger mice) (Figure 60). Total white blood cells (corresponding 60A) decreased with the addition of FS or FLC in the diet but were not significant. However, when looking at differences between cells, especially macrophage levels, levels were significantly reduced by both flaxseed and SDG lignan diets (FIG. 60B).

  Flaxseed and its SDG-rich lignan component reduced asbestos-induced inflammation (old mice) (FIG. 61). Aged mice exposed to abdominal asbestos (FIG. 61A) show 3,000,000 WBC / mL abdominal lavage fluid (10 times more) compared to just 300,000 cells / mL, More sensitive. The results showed that neutrophils of inflammatory cells (FIG. 61B) and macrophages (FIG. 61C) were both significantly more aged in older mice than in younger mice.

  Flaxseed lignan extract rich in SDG (administered in dietary formulations) reduces asbestos inflammation in aged mice (FIG. 62). Male NF2 (129SV) (+/−) mice were injected with 400 μg asbestos on day 0 (intraperitoneal). Mice were started on the test diet (0% FS or 10% FLC) in the week prior to asbestos exposure (day -7) and sacrificed on day 3 after asbestos exposure. Abdominal lavage (AL) was performed with 5 mL 1 × PBS (1 mL of abdominal lavage fluid was centrifuged and the supernatant was frozen). Plasma was collected and frozen at -80 ° C. Cells in the lavage fluid were evaluated and showed that a diet rich in SDG significantly reduced total WBC and neutrophils, macrophages and eosinophils.

  Flaxseed lignan extract rich in SDG (administered in dietary formulations) reduces asbestos inflammatory cytokine secretion and nitrosative stress in aged mice (FIG. 63). Male NF2 (129SV) (+/−) mice were injected with 400 μg asbestos on day 0 (intraperitoneal). Mice were started on the test diet (0% FS or 10% FLC) in the week prior to asbestos exposure (day 7) and sacrificed on day 3 after asbestos exposure. Abdominal lavage (AL) was performed with 5 mL 1 × PBS (1 mL of abdominal lavage fluid was centrifuged and the supernatant was frozen). Plasma was collected and frozen at -80 ° C. A diet rich in SDG significantly reduced the levels of cytokines IL1β and TNFα and nitrite.

  Briefly, these in vivo experiments using NF2 mice indicate the following: (1) Mice fed a diet rich in SDG significantly reduced abdominal inflammation as measured by abdominal lavage fluid WBC; (2) A diet rich in SDG reduced the number of neutrophils in the abdominal lavage fluid (3) levels of pro-inflammatory cytokines, IL-1β and TNFα were reduced in mice fed a diet rich in SDG; and (4) mice fed a diet rich in SDG were abdominal lavage nitrite Of nitrosation stress induced by exposure to asbestos fibers.

  These studies in these examples demonstrate the chemoprotective activity of SDG. To assess the induction of phase 2 enzymes in the oral epithelium and reduction of oxidative stress by SDG, a larger biomarker survey is conducted. Additional measurements are made that measure oxidative stress and inflammation after daily administration of oral SDG in subjects exposed to carcinogens, eg, previous or current smokers. These measurements include plasma oxidative stress measurement (plasma malondialdehyde) and pro-inflammatory stress markers (eg, IL-6, IL-1α, IL1β, TNF-α, C-reactive protein, F2-isoprostane). ) Measurement.

  While particular features of the invention have been illustrated and described herein, many modifications, substitutions, changes and equivalents will occur to those skilled in the art. It is therefore to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims (89)

  A method of protecting a biomolecule, cell, or tissue from radiation damage in a subject in need thereof, comprising administering to said subject an effective amount of at least one bioactive component, said bioactive component comprising: A method comprising secoisolarisiresinol diglucoside (SDG), secoisolarisiresinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or combinations thereof .   The method of claim 1, wherein the subject is a subject that will be exposed to radiation.   The method of claim 1, wherein the subject is a subject that has been exposed to radiation.   2. The method of claim 1, wherein the subject has or will be exposed to radiation as part of a treatment procedure.   5. The method of claim 4, wherein the subject is a cancer patient undergoing or who will receive radiation therapy.   6. The method of claim 5, wherein the cancer patient is a lung cancer patient.   2. The method of claim 1, wherein the subject has or will be exposed to radiation as part of a diagnostic procedure.   8. The method of claim 7, wherein the diagnostic procedure is a dental or bone x-ray examination.   The method of claim 7, wherein the diagnostic procedure is a PET or CT scan.   The method of claim 1, wherein the subject has been accidentally exposed to radiation.   The method of claim 1, wherein the subject has or will be exposed to radiation as part of their occupation.   The method of claim 11, wherein the occupation is a laboratory technician.   The method of claim 1, wherein the subject has been exposed to radon.   The method of claim 1, wherein the subject has been exposed to radiation as a result of terrorism.   A method of protecting biomolecules, cells or tissues from carcinogen-induced damage, malignant transformation and cancer development in a subject in need thereof, wherein an effective amount of at least one bioactive ingredient is administered to said subject The bioactive component is secoisolaricillinol diglucoside (SDG), secoisolaricillinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, steric forms thereof A method involving isomers, or combinations thereof.   16. The method of claim 15, wherein the subject is a subject that will be exposed to one or more carcinogens.   16. The method of claim 15, wherein the subject is a subject that has been exposed to one or more carcinogens.   The method of claim 15, wherein the subject has cancer.   16. The method of claim 15, wherein the subject has or will be exposed to accidental exposure to one or more carcinogens.   16. The method of claim 15, wherein the subject has or will be exposed to one or more carcinogens or chemical war toxicants as a result of terrorist actions.   16. The method of claim 15, wherein the subject has or will be exposed to one or more carcinogens as a result of addiction.   The method of claim 21, wherein the addiction is smoking.   16. The method of claim 15, wherein the subject has or will be exposed to one or more carcinogens as a result of their occupation.   24. The method of claim 23, wherein the subject occupation is a laboratory technician.   A method of protecting a biomolecule, cell, or tissue from damage by hypochlorite ions in a subject in need thereof, comprising administering to said subject an effective amount of at least one bioactive component, The bioactive component is secoisolaricillinol diglucoside (SDG), secoisolaricillinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or these A method involving a combination of   26. A method according to any one of claims 1 to 25, wherein the biomolecule is a nucleic acid.   26. A method according to any one of claims 1 to 25, wherein the biomolecule is a protein or lipid.   The method according to any one of claims 1 to 25, wherein the physiologically active ingredient is (S, S) -SDG.   The method according to any one of claims 1 to 25, wherein the physiologically active ingredient is (R, R) -SDG.   26. The method according to any one of claims 1 to 25, wherein the bioactive component is a synthetic SDG.   26. The method of any one of claims 1-25, wherein the bioactive component is an SDG analog.   26. The method of any one of claims 1-25, wherein the bioactive ingredient is administered in an edible composition.   26. The method of any one of claims 1-25, wherein the administering step comprises administering orally.   26. The method of any one of claims 1-25, wherein the bioactive ingredient is SDG at a concentration of about 1 nanomolar (nM) to about 1 molar (M).   35. The method of claim 34, wherein the concentration of SDG is from about 25 [mu] M to about 250 [mu] M.   26. The method of any one of claims 1-25, wherein the subject is a human subject.   A method of treating or preventing radiation damage in a subject that has been or will be exposed to radiation, comprising administering to said subject an effective amount of at least one bioactive component, The physiologically active ingredient is secoisolaricillinol diglucoside (SDG), secoisolaricillinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or these A method involving combinations.   38. The method of claim 37, wherein the subject has or will be exposed to radiation as part of a treatment procedure.   40. The method of claim 38, wherein the subject is a cancer patient undergoing or will receive radiation therapy.   40. The method of claim 39, wherein the cancer patient is a lung cancer patient.   38. The method of claim 37, wherein the subject has or will be exposed to radiation as part of a diagnostic procedure.   42. The method of claim 41, wherein the diagnostic procedure is a dental or bone x-ray examination.   42. The method of claim 41, wherein the diagnostic procedure is a PET or CT scan.   38. The method of claim 37, wherein the subject has been accidentally exposed to radiation.   38. The method of claim 37, wherein the subject will be exposed to have been exposed to radiation as part of their occupation.   46. The method of claim 45, wherein the subject occupation is a laboratory technician.   38. The method of claim 37, wherein the subject has been exposed to radon.   38. The method of claim 37, wherein the subject has been exposed to radiation as a result of terrorism.   A method of treating or preventing carcinogen-induced damage, malignant transformation or cancer development in a subject who has been or will be exposed to one or more carcinogens, comprising an effective amount At least one physiologically active ingredient of the above, wherein the physiologically active ingredient is secoisolaricillesinol diglucoside (SDG), secoisolarisiresinol (SECO), enterodiol (ED), enterolactone (EL), a method comprising these analogs, stereoisomers thereof, or combinations thereof.   50. The method of claim 49, wherein the carcinogen-induced injury, malignant transformation or cancer development is cancer.   51. The method of claim 50, wherein the cancer is lung cancer.   51. The method of claim 50, wherein the cancer is malignant mesothelioma.   51. The method of claim 50, wherein the subject has not developed the cancer.   50. The method of claim 49, wherein the subject has or will be exposed to accidental exposure to one or more carcinogens.   50. The method of claim 49, wherein the subject has or will be exposed to the one or more carcinogens or chemical war toxicants as a result of terrorist actions.   50. The method of claim 49, wherein the subject has or will be exposed to one or more carcinogens as a result of addiction.   57. The method of claim 56, wherein the addiction is smoking.   50. The method of claim 49, wherein the subject has or will be exposed to one or more carcinogens as a result of their occupation.   59. The method of claim 58, wherein the subject occupation is a laboratory technician.   50. The method of claim 49, wherein the one or more carcinogens are asbestos.   50. The method of claim 49, wherein the one or more carcinogens are tobacco smoke.   50. The method of claim 49, wherein the subject is a smoker.   50. The method of claim 49, wherein the subject is a former smoker.   50. The method of claim 49, wherein the subject is a non-smoker exposed to sidestream smoke.   A method of treating or preventing hypochlorite ion-induced damage in a subject who has been or will be exposed to hypochlorite ions, comprising an effective amount of at least one bioactive ingredient. Administration to the subject, wherein the bioactive component is secoisolarisiresinol diglucoside (SDG), secoisolarisiresinol (SECO), enterodiol (ED), enterolactone (EL), and analogs thereof , These stereoisomers, or a combination thereof.   The method according to any one of claims 37 to 65, wherein the physiologically active ingredient is (S, S) -SDG.   66. The method according to any one of claims 37 to 65, wherein the bioactive component is (R, R) -SDG.   26. The method according to any one of claims 1 to 25, wherein the bioactive component is a synthetic SDG.   66. The method of any one of claims 37 to 65, wherein the bioactive component is an SDG analog.   66. The method of any one of claims 37 to 65, wherein the bioactive ingredient is administered in an edible composition.   66. The method of any one of claims 37 to 65, wherein the administering step comprises administering orally.   66. The method of any one of claims 37 to 65, wherein the bioactive component is SDG at a concentration of about 1 nanomolar (nM) to about 1 molar (M).   73. The method of claim 72, wherein the concentration of SDG is from about 25 [mu] M to about 250 [mu] M.   66. The method of any one of claims 37 to 65, wherein the subject is a human subject.   A method of protecting a biomolecule, cell, or tissue from radiation damage comprising contacting the biomolecule, cell, or tissue with an effective amount of at least one bioactive component, wherein the bioactive component is A method comprising silesinol diglucoside (SDG), secoisolaricillinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or combinations thereof.   A method of protecting a biomolecule, cell or tissue from carcinogen-induced damage comprising contacting said biomolecule, cell or tissue with an effective amount of at least one bioactive component, said bioactivity Ingredients include secoisolaricylesinol diglucoside (SDG), secoisolarisiresinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or combinations thereof Including methods.   A method of protecting a biomolecule, cell, or tissue from damage by hypochlorite ions, comprising contacting the biomolecule, cell, or tissue with an effective amount of at least one bioactive component, The active ingredient is secoisolarisiresinol diglucoside (SDG), secoisolarisiresinol (SECO), enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomers thereof, or combinations thereof Including methods.   78. A method according to any one of claims 75 to 77, wherein the biomolecule is a nucleic acid.   78. A method according to any one of claims 75 to 77, wherein the biomolecule is a protein or lipid.   76. The method according to any one of claims 73 to 75, wherein the physiologically active ingredient is (S, S) -SDG.   78. The method according to any one of claims 75 to 77, wherein the physiologically active ingredient is (R, R) -SDG.   78. The method according to any one of claims 75 to 77, wherein the physiologically active ingredient is synthetic SDG.   78. The method of any one of claims 75 to 77, wherein the bioactive component is an SDG analog.   78. The method of any one of claims 75-77, wherein the bioactive component is SDG at a concentration of about 1 nanomolar (nM) to about 1 molar (M).   85. The method of claim 84, wherein the concentration of SDG is from about 25 [mu] M to about 250 [mu] M.   78. The method of any one of claims 75 to 77, wherein the cell or tissue is a cancer cell or tissue.   90. The method of claim 86, wherein the cancer is lung cancer.   90. The method of claim 86, wherein the cancer is malignant mesothelioma.   78. The method of any one of claims 75 to 77, wherein the biomolecule, cell or tissue is a human biomolecule, cell or tissue.