Free Radicals and Antioxidants i

Free Radicals and Antioxidants in Obstetrics and Gynecology Prof.Surendra Nath Panda, M.S & Dr.Sasmita Das, M.B.B.S. Department of of Obstetrics & Gynaecology M.K.C.G.Medical College Berhampur, Orissa, 760004, India Introduction There was an era when people were worried about the disease processes and its treatment. But off late, tremendous efforts are being made to find out the ways of prevention of important diseases. The important research studies are rightly to be repeated with other populations, but nevertheless give an opportunity to consider the integrated actions of the known reducing (Antioxidant) systems. Inadequacy of certain internally produced reducing systems has been postulated as being at the core of some disease processes. It is believed that life has originated from basic chemicals by free radical reaction, largely initialled by ionising radiation from sun. Paradoxically the same reactions creating life may also be responsible for many diseases, ageing and death. 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 2 Free Radicals (FRs) • These are highly reactive chemical entities that have a single unpaired electron in their outer most orbit. • Under certain conditions can be highly toxic to the cells. • Generally unstable and try to become stable, either by accepting or donating an electron. • Therefore if two FRs react, they neutralise each other. • However, if the FRs react with stable molecules, there is generation of more free radicals. • This characteristic enables the FRs to participate in auto catalytic chain reactions, – Molecules with which they react are themselves converted to free radicals to propagate the chain of damages. 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 3 Reactive Oxygen Species (ROS): These are free radicals derived initially from oxygen. But as they do not contain unpaired electrons in their outermost orbit, they do not qualify as free radicals and so are referred to separately as ROS. Eg.- H2O2, HOCL, NO. Free radicals are formed in side our body by both PHYSIOLOGICAL (Natural) and PATHOLOGICAL stimuli : 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 4 Physiological Stimuli that Form FRs • Normal respiration – – – – – O2 – Superoxide, H2O2 – Hydrogen Peroxide HOCL – Hypochlorous acid NO – Nitric Oxide • Transition metals present inside our body when are in free form behave as free radicals. Fe2+, Cu+ • Body cells– Endothelium (NO3 – Nitric Oxide, NO2 – Nitrous Oxide), – Macrophages (NO2) – Neurones (ONOOH – Peroxy nitrite). • • • • Ageing Phagocytosis or biogenetics Oxidation of foods and endogenous compounds. Transportation of substances for energy production. 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 5 Pathological Stimuli that Form FRs • Radiation Breaks the water inside our body: H2O =H+ + OH• Metabolism of drugs  CCl3 • Transition Metals  Cu+, Fe2+ • Ultraviolet rays • Emotional stress 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 6 Actions of FRs Mechanism of Action: • They act on the cell membranes and membranes of different organelles of cells and cause cell injury and death by oxidative reactions. • So FRs are also called OXIDANTS. 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 7 Actions of FRs • FRs cause lipid peroxidation. – The PUFA of cell membrane are more vulnerable for this injury. By lipid peroxidation FR increases the permeability of cells, leading to calcium influx and altered PH of the cell. • FRs alter the enzyme and receptor proteins. – FRs cause – cross-linking of proteins and fragmentation of protein strands, oxidation of amino acids like cystene, Methionine. – These alterations in the enzymes and receptoors inside the cell lead to abnormal cell behaviour. 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 8 Actions of FRs • FRs cause fracturing on the cell nucleus resulting in single strand DNA damage. This oxidative injury may be. – Lethal – Leading to cell death and ultimately removed by phagocytosis. – Sub lethal - which may result in • Increased cell permeability. • Toxicity. • Mutation of cells. 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 9 FRs Induce Chain Reaction • During the process of oxidant damage resulting in tissue destruction & degeneration, some electrons may escape oxidation and become FRs • This chain reaction may produce diseases like: – – – – – – – – – Carcinogenesis. Myocardial reperfusion injury. Shock related injury. Arteriosclerosis. Rheumatoid arthritis. Adult respiratory diseases. Diabetes. Obesity. Lipid abnormality. Etc. 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 10 Antioxidants (AOs) • These are substances, which protect us against potentially harmful free radicals derived from oxygen. • To combat the injurious effects of FRs our body has its own system of „in vivo‟ Antioxidants‟. • In normal healthy state a balance is maintained between FRs & AOs • The „AO‟ activity of serum is measured as - % inhibition of lipid peroxidation in a standardised brain homogenate. • Moreover we can as well supplement these from outside (in vitro Antioxidants). 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 11 In Vivo Antioxidants: I.Enzymes Name SOD (Superoxide dismutase) CATALASE Acts against Super oxide H2 O2 Present in cytosol mitochondria. Blood, bone marrow, Mucus membrane Kidney; Liver. membranes of lipids, Haemoglobin and erythrocytes GOP (Glutathion peroxidase) H2 O2, lipid peroxidation 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 12 In Vivo Antioxidants: II. Preventive AOs These are binding proteins. They keep the free ions of plasma in a binding form, so prevent oxidation injury. Eg.-Transferin for Fe, Ceruloplasmin for Cu III. Scavenger AOs Also called chain breaking enzymes, they break the catalytic chain propagated by FRs. 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 13 In Vivo Antioxidants: - Source I. In the form of Medicines: Vitamin A, C & E, Cystine, Glutathion, Melthionine, Bioflavines, Se, Zn. II. Food sources:  Green & yellow vegetables  Herbs: -Turmeric, Garlic, Grape, Tea, Berries, Carrot, Spinach, Broccoli,  Red Meat, Kidney, Liver & Lipoic Acid 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 14 Oxidative Stress: • Under normal conditions body maintains an equilibrium between its own FR‟s and Antioxidants. • When this equilibrium breaks, a state called oxidation stress arises with in, due to  FR formation or  AO system. 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 15 AO Status in Normal Pregnancy: • There is increased need for AOs as there is increased production of FRs due to – Pregnancy being a stressful condition. – Because of the rapidly growing foetus there is increased cellular activity. • Thus AO activity during normal pregnancy progressively increases as demonstrated by –  Serum tocopherol. –  Activity of GOP. –  Serum ceruloplasmin & transferring level. • However, there is no evidence to suggest the need for administration of „AOs‟ during normal pregnancy but drugs that lead to „FR‟ formation must be avoided. 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 16 AOs & Diabetic Embryopathy: • Oxidative stress has been suggested to contribute to the increased risk of foetal malformations in poorly controlled diabetics. – There are reports of  lipid peroxidation in cell membranes in diabetic pregnancies – Periods of maternal hyperglycaemia & hypoglycemia may cause marked changes in the availability of glucose to the foetus. – Also  conc. of lipids, notably the ketone bodies and branched chain amino acids in the maternal circulation contribute to altered nutrition for the embryo Leading to  FR conc. & foetal malformation in embryo. 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 17 AOs & Diabetic Embryopathy: • During later part of pregnancy –  load of glucose in the mitochondria may accelerate the flow of electrons through respiratory chain including mitochondrial leakage of free radicals. – This leads to  production of FR in embryonic tissues to cause congenital malformations. • Thus maintenance of normal concentration of metabolitetes of all nutrient class may be important for prevention of adverse foetal outcome. • But the question is “ will dietary supplements alone hold the key to the future in diabetic embryopathy”? • However, maintenance of blood glucose level at euglycemic level is always important for prevention of Diabetic embryopathy . 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 18 AOs & Down Syndrome: • Free radicals being the hallmark of aging, are greatly increased with maternal age. • So FRs play a role in pathogenesis of Down‟s syndrome. • Administration of AOs may help in preventing this disease. 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 19 FRs & AOs in PIH : • Increased activity of free radicals promote maternal uterine vascular malformations. – FRs are promoters of maternal vasoconstriction. • O2 , H2 O2 & NO2 in combinations – Inactivate the NO (a vasorelaxant) – Causes  PG synthatase activity. – Produce peroxynitrate, a potent oxidant • leading to the subsequent development of PIH (See also notes page). • Evidence-. – Lipid peroxide in pre-eclamptic placenta is about 1.8 times higher in comparison to normal placenta. – Vit. E is less in serum of PIH patients. – Severity of hypertension has been found to be inversely proportional to concentration of Vit. E. 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 20 FRs & AOs in Preterm Delivery: • Sub clinical chorioamnionitis leads to liberation of bacterial antitoxins (polysaccharide in nature) and inflammatory cytokines. • These cause stimulation of inflammatory cells and cells of CX. which in turn produce NO2. • Evidence: There is  N2O in vaginal samples of females having preterm delivery. • So antioxidants may play a part in the treatment of preterm delivery. 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 21 FRs & AOs in New Born Babies: • In recent years experimental and clinical data have provided compelling evidences for involvement of oxygen derived „FRs‟ in disorders of prematurity. – – – – Chronic lung disease Retinopathy Intra ventricular haemorrhages. Necrotising Enterocolitis: - FRs cause microvascular injury and  cell permeability leading to necrotising enterocolitis. 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 22 FRs & AOs in New Born Babies: • PDA: –  Hypoxanthine radical in NBB leads to  formation of PGE2 and causes PDA. – Studies suggest that kwashiorkor babies have FRs in their body . – „AO‟ in Kwashiorkor babies are due to deficiency of Vit. A, E, C, Inositol & Selinium. • Kwashiorkor: - • Ideally the future treatment of Neonates with all these disease should include AO therapy. 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 23 Carcinogenesis: • Basics of cancer formation: – A normal cell can undergo malignant transformation in presence of procarcinogens and carcinogens. – However, in early stages, it can revert back to normal cell when detected and corrected by our body immune system. – Without immune system detection a normal cell is converted to malignant cell in stages – • Initiation  Promotion  Progression 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 24 FRs in Carcinogenesis: • Free radicals are formed from stimulants like – Radiation, Xenobiotics, Inflammatory cells, Respiration etc, which act on cellular targets to cause oxidant DNA damage in form of mutagenesis & clostogenesis, which cause: – Initiation of carcinogenesis by• Activation of protooncogens. • Inactivation or loss of tumour suppresser genes. • Normal cell becomes initiated cell. – Procarcinogens are metabolically activated by FRs, which cause promotion and progression of these initiated cells to cancer – Ultimately carcinogenesis sets in 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 25 FRs & AOs in Carcinogenesis: FRs  Activation of procarcinogens & Carcinogens Genetic Damage Normal cell  Initiation    Repair by AOs Initiated Cell  Promotion FRs  - Tumour promoters - Spontaneous    Repair by AOs Premalignant Cell    Repair by AOs  Progression    FRs  - Dysregulation - Greater Cell autonomy - Reduced Growth Factor dependence Malignant Cell  CLINICAL CANCER 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 26 AOs in Cancer Prevention: • DEFENCE: - enzymes with antioxidant property cause first line of defence by: – Protecting the lipids and enzymes against oxidation Retarding the generation of free radicals  Creating a balance of „AOs‟ against FRs in the body  PREVENTS : • • • • Cell pathology. Metabolic disturbances. Changes in cell permeability. Eormation of toxic products.  Prevention of initiation of Carcinogenesis 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 27 AOs in Cancer Prevention: • INTERVENTION at promotion & progression stages: – Local deactivation of genotoxins responsible for further nuclear mutations – Inactivation of tumour promoters eg.- activation of granulocytes. – Simulate oxygen. – Maintenance of proper function of gap junction communication. – Maintenance of physical stability of membrane & also within cells. 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 28 AOs in Cancer Prevention: • Majority of epidemiological data suggest supplementation with antioxidants- Vit.- A, E, C; beta carotene & selenium, decreases the incidence of various cancers. 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 29 AOs in Cancer Prevention: RECOMMENDED FOR CARCINOMA PREVENTION (Federation of Obstetric & Gynaecological Societies of India) Antioxidant Vitamin „A‟ RDA 5000 IU Recommended Possible Dose Toxicity Level 12,500 IU Features Causing Req. Chronic intake Smoking of 125,000 IU Vitamin „E‟ Vitamin „C‟ 10-20 IU 60 mg 200-800 IU 1000 mg >1,200 IU Negligible / 12Gms High PUFA intake, Smoking Stress, OCP,Smoking Selenium None 50-200 mg >200mg Aging, High PUFA intake, Smoking, Heavy metals 30 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das FRs & AOs in Infertility - Male • Various studies have suggested Conc. of Malon –di-Aldehyde (MDA) is inversely proportional to fertility in case of males. • In asthenospermic and oligoasthenospermic males there is increased serum concentration of MDA. • Even in normospermic males if there is  concentration of MDA there is reduced fertility. • Addition of vitamin E causes decreased concentration of MDA and improves fertility. 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 31 FRs & AOs in Infertility - Female • FRs cause – Short luteal phase and LPDS. – In IVF arrest the cell growth (div) at 2, 4, 8 cell stages – Hamper the regulation of corpus luteum. • Addition of „AOs‟ improves the results. 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 32 CONCLUSION Traffic lights are to cars, what „AOs‟ are to the cells. Oxidant generation is a part of human life. Every O2 atom we breath in ,is converted to water inside our body by addition of four electrons sequentially. When four electrons are added three „FRs‟- O2, H2 O2 & OH are formed along with water. So where there is life there are oxidants. 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 33 CONCLUSION But when produced in excess, they can cause any disease. So our concern should be to  FRs in systemic circulation. However, careful use of „AOs‟ and newer and more accurate methods to measure oxidant generation in humans will go a long way to find out the exact contribution of oxidants in disease processes and the role of „AOs‟ to prevent it. 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 34 THANK YOU 30/08/2002 Antioxidants in Obstetrics and Gynaecology - Prof.S.N.Panda & Dr.Sasmita Das 35