Antioxidants : Past And Present

The role of oxidative stress is well recognized now in numerous pathological states, where antioxidants can play very important role. Recent studies provide evidences that low levels of antioxidants are associated with increased risk for many pathological states and that increased intake appears to be protective. However, there are conflicting studies also, that question the rationale for antioxidant supplementation in these conditions by suggesting detrimental effects to the health. Suicidal oxidative stress produced by some of the antioxidants under certain circumstances remains the major cause of concern ,as many antioxidants can act as pro-oxidants in certain conditions. Moreover, at present there are number of unanswered questions associated with practical application of antioxidant therapy like appropriate timing of administration, dose & duration of therapy, which still need to be determined. Inspite of these concerns, antioxidant therapy has gained an important status presently in the prevention and treatment of various pathological conditions. However, under the shadow of these concerns, the supplementation of natural dietary antioxidant resources seems to be a safe and effective approach currently.

KEY WORDS : Oxidative stress, antioxidants, present, past, status.

The free radical can be defined as a chemical species, an atom or a molecule that has one or more unpaired electrons in its valance shell and is capable of existing independently. As free radical contains an odd number of electrons which makes it unstable, short lived and highly reactive, therefore it can react quickly with other compounds in order to capture the needed electron to gain stability. Generally, free radical attacks the nearest stable molecule “stealing” its electron. When the attacked molecule looses its electron, it becomes a free radical itself, beginning a chain reaction cascade resulting in disruption of a living cell^1,2. Most common radical derivatives of oxygen like superoxide free radical anion (O₂^·‑), hydroxyl free radical (OH^·), lipid peroxyl (LO^·), lipid alkoxyl (LOO^·) and lipid peroxide (LOOH) as well as non-radical derivatives such as hydrogen peroxide (H₂O₂) and singlet oxygen (¹O₂) are collectively known as reactive oxygen species (ROS)². These free radicals/reactive oxygen species are produced mainly from two important sources^1,2 in the biological system i.e. cellular metabolism and environmental sources.

Table-I: SOURCES OF THE REACTIVE OXYGEN SPECIES IN BIOLOGICAL SYESTM ^{1 , 2}

SOURCES	EXAMPLES
Cellular Metabolism	· Mitochondrial electron transport · Endoplasmic reticulum oxidation · Enzymatic activity like : NADPH oxidase, Xanthine oxidase, Monoamine oxidase, Tyrosine hydroxylase, L-amino oxidase, Diamine oxidase, Glycolate oxidase, α-hydroxy acid oxidase, L –gluconolactone oxidase. · Prostaglandin synthesis. · Autooxidation of adrenaline, Thiol, Ascorbic acid · Reduced riboflavin, FMNH2 FADH2 · Nitric oxide sythatase · Stimulated neutrophils · Activated phagocytic cells · Reperfusion injury. · Cytochrome P450.
Environmental	· Drugs: Halothene, Paracetamol, Bleomycine, Doxorubicin, Metrenidazole, Ethanol. ·CCl4. · Pesticides ·Transition Metals (Cu, Fe) ·Tabacco Smoke · Alcohol · Radiations · X-ray by water radiolysis · Light by photoionization · High Temperature

 

Free radical and reactive oxygen species production in the animal cell is inevitable.

Normally, there is an equilibrium between a free radical/reactive oxygen species formation and endogenous antioxidant defense mechanisms, but if this balance is disturbed, it can produce oxidative stress^{2, 3}. This state of oxidative stress can result in injury to all the important cellular components like proteins, DNA and membrane lipids which can cause cell death.

In recent years increasing experimental and clinical data has provided compelling evidences for the involvement of FR/ROS in large number of pathophysiological states^1,2,4.

This has led to increasing curiosity and interest among the scientists and researchers globally to evaluate potential benefits from antioxidant therapy. Presently studies provide evidences that low levels of antioxidants are associated with increased risk for many pathological states and that increased in take appears to be protective^5-11.

However, there are conflicting reports also which question the rationale for antioxidant supplementation by suggesting them to be ineffective^12-20 or in some cases detrimental^21,22 to the health. Moreover, there are few concerns and some unsolved questions, which still remain to be determined in the practical application of antioxidant therapy. Therefore, an attempt to understand, the mechanism of antioxidants and their past and present status is made in the present article.

 

ANTIOXIDANTS: are the compounds of exogenous or endogenous in nature which either prevent the generation of toxic oxidants or intercept any that are generated and inactivate them and thereby block the chain propagation reaction produced by these oxidants²³. Halliwell and Gutteridge²⁴ have defined antioxidants as substances which are present in low concentrations and can significantly delay or inhibit oxidation of oxidisable substrates.

Types of antioxidant defenses²⁵

1. Primary or chain breaking antioxidants (scavenger antioxidants) : these antioxidants can neutralize free radicals by donating one of their own electron, ending the electron “stealing” reaction. The resultant antioxidants which become free radicals, because of one electron left in their outer shell, are relatively safe, stable and in normal circumstances insufficiently reactive to initiate any toxic effect e.g. α-tocopherol

2. Secondary or preventive antioxidants: act through numerous possible mechanisms like a) sequestration of transition metal ions which are not allowed to participate in metal catalyzed reactions. b) removal of peroxides by catalases and glutathione peroxidase, that can react with transition metal ions to produce ROS. c) removal of ROS etc.

3. Tertiary antioxidant defenses: are the repair processes, which remove damaged biomolecules before they can accumulate and before their presence results in altered cell metabolism and viability. Damaged DNA repaired by enzyme methionine sulphaoxide reductase, oxidized proteins are removed by proteolytic enzyme system and oxidized lipids are acted upon by lipases, peroxidases and acyl transferases are some of the examples.

Table 1, 2 & 3 shows sub-classification of various known antioxidants and their mechanism of defense against ROS in biological system.

TABLE – 1 : ANTIOXIDANTS IN BIOLOGICAL SYSTEM

Antioxidants	Mechanisms	ref’s.
(a) Enzymatic
Superoxide dismutase (SOD)	+ 1e O2. – →+ 2H → H2O2 SOD (detoxifies superoxide anion)	1,23,26
Catalase	2H2O2 → Catalase → 2H2O + O2 (detoxifies hydrogen peroxide)	1,23,26
Glutathione Peroxidase (GPX)	2GSH + H2O2 → GPX → GSSG + 2H2O (detoxifies hydrogen peroxide & xenobiotics biotransformation)	1,2
Glutathione Reductase (GRD)	GSSG + NADPH + H+ → GRD → 2GSH + NADP+ (regeneration of reduced glutathione and xenobiotic biotransformation)	1,2
Glucose – 6 Phosphate ehydrogenase (G6PD)	·G6PD+NADP+→NADPH+H++6Phosphoglucolactone ·NADPH + H+ + Oxidised (GSSG)→ NADP++Reduced (GSH) Oxidatively →Reduced (GSH)→ Healthy RBC damaged membrane R.B.C membrane (prevents oxidative damage to RBC membrane)	2
Cytochrome Oxidase System	detoxifies 95-99% of O2 in cell.	23
Peroxidase	detoxifies hydrogen peroxide	23

O₂^{. –}(superoxide anion), H₂O₂(hydrogen peroxide), GSH (reduced glutathione), GSSG (oxidised glutathione)

TABLE – 2 : ANTIOXIDANTS IN BIOLOGICAL SYSTEM

 

Antioxidants	Mechanisms	ref’s.
(b) Non-enzymatic
(i) Nutrient
Carotenoid (β-Carotene)	· Chain breaking antioxidant prevents propagation of lipid peroxidation by trapping peroxide free radicals at low partial pressure in its conjugated alkyl structure. · Scavenger of singlet oxygen and superoxide free radical. · Also complements Vitamin-E, which is effective at higher O2 concentration.	2
α-Tocoferol	· Prevents lipid peroxidation. LOO.+ α-tocopherol-OHÕLOOH+α-tocopherol-O. which is relatively safe and stable. · Stabilizes cell membrane. · Major natural protective agent against ROS. · Requires Vitamin-C and selenium for complete reaction.	2
Ascorbic acid	· Prevents lipid peroxidation both in plasma and inside the cell. TOCO.+Vit.C(GSH)ÕRegeneratedTOCOH+Vit.C(GSSG) · Protects LDL against peroxidative damage. · Inactivates OH., O2.-& 1O2.	2
(ii) METABOLIC
Glutathione	· Metabolism of xenobiotics. · H2O2 detoxification	23
Uric acid	· Scavenger of 1O2 & OH.. · Binds Fe & Cu ion in the form that they do not accelerate free radical reactions	23
Albumin	·Inhibits lipid peroxidation.	23
Transferrin/ lactoferrin/ ceruloplasmin	·Binds to circulating Fe & Cu and therefore prevent Fe & Cu dependent lipid peroxidation.	23
Ferritin	· Binds tissue Fe.	23
Hepatoglobin	· Binds free Hb/heme, therefore prevents release of Fe and indirectly prevents Fe dependent lipid peroxidation.	23
Cysteine	· Scavenger of O2.-& OH.	23
Bilurubin	· Scavenger of O2.-& OH.	23

LOO^.(lipid peroxyl radical), α-tocopherol-O^.(tocopheroxyl radical),α-tocopherol-OH(hydroxyl tocopherol), LOOH (lipid hydroperoxide), TOCO^. (tocopheroxyl radical), Vit.C-GSH(reduced), Vit.C-GSSG (oxidised), TOCHOH (regenerated tocopherol),OH^.(hydroxyl radical), ¹O₂(singlet oxygen), O₂^.–(superoxide anion).

TABLE – 3 : ANTIOXIDANTS IN BIOLOGICAL SYSTEM

(c) Natural antioxidants27-30 (derived from natural sources) Flavonoids, coumarins, phenolic acid, linolenic fatty acid, Omega – 3 fatty acid, α-tocoferol, β-carotene and ascorbic acid etc. (d) Pharmocological/synthetic antioxidants23

Probucol, Xanthine oxidase inhibitor (allopurinol, folic acid) Superoxide dismutase Catalases NADPH inhibitors (adenosine, calcium channel blockers) Antioxidants raising endogenous glutathione peroxidase activity (glutathione, acetylcysteine) Inhibitors of iron redox cycling (deferoxmine, apotransferin, ceruloplasmin) Non-steroidal anti-inflammatory agents31 Oral anti-diabetic agents32-34 like (metformin, gliclazide & troglitazone) Statins35,36 (atorvastatin, simvastatin, pravastatin & rosuvastatin) Omeprazole37

ANTIOXIDANTS-PRESENT STATUS

It is now well recognized that oxidative stress participates in the pathogenesis of various diseases/conditions^1,2,4. Present studies provide mixed evidences, as some of the reports suggest increased intake of antioxidants to be protective and other suggest their supplementation to be ineffective in various pathological conditions like cardiovascular diseases, cancer, neurodegenerative diseases, cataract ,ageing, liver injury and complications of type-2 diabetes-mellitus. Whereas, promising/encouraging results are indicated in inflammatory joint conditions, ionizing radiation induced cell damage, sterility, septic shock and hyperlipidemia etc. However, there are conflicting studies also, which suggests vitamin antioxidants may not only be ineffective^12-20 but can also produce deleterious effects^21,22 to the health. In addition, there are few concerns and unsolved questions regarding the practical application of antioxidant therapy. Inspite of this, antioxidants have gained an important status presently in prevention and treatment of various diseases.

1.Cardiovascular diseases (CVD): endothelial dysfunction has relevance to the pathogenesis, progression and prognosis of a wide spectrum of cardiovascular diseases³⁸. The assumption that oxidation of LDL, loss of nitric oxide and the vascular inflammatory response modulated by oxidative stress mediates endothelial dysfunction would implicate a potential for antioxidant therapies to ameliorate endothelial dysfunction. This assumption is supported by one of the study³⁹ done in LDL receptor deficient mice, which showed vitamin-E supplementation can revert these changes. In humans antioxidant vitamins by potentiating endothelial nitric oxide levels^40-42 as well as by inhibiting vascular inflammation⁴³, lipid peroxidation⁴³, platelet aggregation⁴³ and oxidation of LDL^43-46 can also contribute to prevent endothelial dysfunction. Additionally antioxidants may favourably influence plaque stability⁴⁷. Some recent studies provide direct evidences that antioxidant vitamins can reverse endothelial dysfunction induced by methionine⁴⁸ as well as can restore endothelial function in hyperlipidemic children⁴⁹ and young smokers⁵⁰. Allopurinol, (xanthine oxidase inhibitor) a potential antioxidant has been shown to reverse endothelial dysfunction in heavy smokers⁵¹, type-2 diabetics with mild hypertension⁵² and in patients of chronic heart failure⁵³. The Secondary Prevention with Antioxidants of Cardiovascular disease in End-stage renal disease study (SPACE)⁵, the Cambridge Heart Antioxidant Study (CHAOS)⁶ and Transplant Associated Arteriosclerosis Study (TAS)⁷ showed positive results by indicating significant decrease in primary end points in the form of various cardiovascular events and deaths. In the most recent study, Antioxidant Supplementation in Atherosclerosis Prevention (ASAP)⁸, combination of natural antioxidant with ascorbic acid resulted in significant increase in plasma levels of antioxidants and ascorbic acid. Significant decrease in the rate of progression of carotid intimal medial thickness was also observed in the treatment group. Moreover, no deleterious effects were observed with this therapy in the patients under study, thereby clearly indicating that antioxidants slow down atheroclerotic progression. In coronary artery disease patients, it is suggested that increasing glutathione-1 peroxidase activity might lower the risk of cardiovascular events⁵⁴. Similarly catalase by enzymatic inactivation of ROS⁵⁵, super-oxide dismutase by regulating the availability of nitric oxide⁵⁶ and selenium by increasing glutathione peroxidase activity⁵⁷, might be protective against cardiovascular events in such patients.

However, the results of recent prospective antioxidant clinical trials^12-17 have been disappointing with regard to primary end point of cardiovascular events, as these trials found no overall protective relation between them. So they have raised considerable doubt about antioxidant related cardiovascular protection in myocardial-infarction, stroke or other CVS events. Not only this vitamin antioxidants may not only be ineffective but can also produce deleterious effects to vascular health^{21, 22}. Beta- carotene supplementation has been suggested to produce significant increase in stroke incidence²¹ and overall cardiovascular deaths²².

2.Carcinogenesis: whether antioxidant therapy has a place in prevention or treatment of carcinogenesis is still unclear. Most debated topic in cancer currently is the question, whether antioxidants are capable of lessening the side effects and can they improve the results of conventional cancer treatments?. Some recent findings support the view that there is a possibility of enhancing therapeutic activity of various chemotherapeutic agents by a concomitant administration of antioxidant drugs e.g. melatonin (antioxidant hormone) with irinotecan or 5-fluorouracil can potentiate the activity of these agents⁵⁸. Similarly vitamin-E with cisplatin produced the neuroprotection⁵⁹ and vitamin-E along with pentoxifylline⁶⁰ was shown to reduce superficial radiation induced fibrosis significantly. Vitamin antioxidant supplementation has been shown to reduce the risk of ovarian⁹, prostate¹⁰ and lung cancer¹¹. Vitamin-A is important for cell differentiation, therefore a protective effect of this vitamin on urological cancer development may also be expected⁶¹. However, a recently published meta-analysis based on seven case control studies and three cohort studies did not reveal increased risk for bladder cancer with diet low in retinol or β-carotene⁶². In agreement to this meta-analysis, prospective studies published recently also found no association between vitamin antioxidant supplementation and bladder cancer^18,19 or colorectal cancer²⁰ risk. However, in one of the recent expert review on cancer care⁶³ high doses of multiple dietary antioxidants as an adjunct to standard therapy to improve tumor response and decrease toxicity as well as lower doses of antioxidants after completion of standard therapy to reduce the risk of recurrence of original tumor has been recommended.

3.Ionizing radiation induced cell damage: antioxidant enzymes such as super-oxide dismutase, catalase, glutathione peroxidase and non-enzymatic antioxidants like selenium⁶⁴ and more recently water soluble vitamin-E (TMG)⁶⁵ have been shown as radioprotectors. Natural nutrient antioxidants also have been suggested to provide protection against ionizing radiation mediated cell damage⁶⁶.

4.Neurodegenerative diseases: one cross section, prospective study found use of vitamin-E and vitamin-C to be associated with reduced prevalence and incidence of Alzheimer’s disease⁶⁷, which was in agreement to one of the previous study⁶⁸. However, it was contrary to the study of Luchsinger JA et al. (2003)⁶⁹ which failed to show any such association.

5.Cataract: melatonin, an antioxidant hormone may protect the eye lens from damaging effect of ultraviolet exposure by reducing oxidative stress⁷⁰ Vitamin-C & E protect cultures of bovine lens epithelium from damaging effect of blue light and UVA light⁷¹. Similarly, alpha lipoic acid decreases iron catalyzed free radical formation in lens epithelium cells⁷². However, recent clinical trials^73,74 indicated antioxidants to provide no benefit or harm on age related cataract and visual acquity loss.

6.Peptic ulcer: gultathione plays a major role in the cytoprotection against ulceration⁷⁵. Various plant derived natural antioxidants like flavonoids act as antiulcerogenic^27,28. A noble antioxidant and antiapoptotic role of omeprazole to block gastric ulcer through scavenging of hydroxyl radical also has been suggested recently³⁷.

7. Inflammatory joint conditions: Many recent studies strongly suggest anti-inflammatory role of superoxide dismutase⁷⁶ and vitamin-E⁷⁷. Vitamin-E has been suggested to inhibit COX enzyme and produce anti-inflammatory effects⁷⁷. One study also provided evidence that NSAID’s help in rheumatoid arthritis by raising circulating superoxide dismutase levels³¹.

8. Liver Injury: Evidence of oxidative stress and depleted levels of antioxidants in patients of liver injury is present⁷⁸. However, non-conclusive results were found in severe alcoholic hepatitis with the use of antioxidant therapy⁷⁹.

9. Sterility: It is suggested that vitamin-E can prevent nonyl-phenol induced oxidative stress in testis of rats, which is known to impair reproduction⁸⁰.

10.Ageing: oxidative stress in the ageing rat heart is reversed by dietary supplementation of alpha lipoic acid⁸¹. However, no recent conclusive evidences are available to support the role of antioxidants in delaying ageing process or influencing total mortality in elderly⁸².

11.Pre-eclampsia: some studies^83,84 have shown promising results to revert or reduce the occurrence of pre-eclampsia in women at increased risk.

12.Acute respiratory distress syndrome and septic shock: reduction in oxidative stress coupled with delivery of NO seems to be a protective strategy for treating ARDS⁸⁵. But whether antioxidant therapy has a place in the treatment of septic shock is unclear. However, few studies^86,87 showed some encouraging results for antioxidant therapy in the treatment of septic shock.

13.Type-2 diabetes mellitus: microangiopathic complications in diabetic patient may be delayed or progress can be retarded by antioxidant therapy⁸⁸. Contrary to this report, one of the most recent study⁸⁹ suggested no significant overall associations between risk of diabetic retinopathy and intake of major dietary antioxidant. Many known anti-diabetic agents like gliclazide³², metformin³³, troglitazone³⁴ are also known to possess antioxidant properties.

14.Hyperlipidemia: Engler et al. (2003)⁴⁹ have advocated the role of vitamin-C and E to improve endothelial dysfunction in hyperlipidemic children. Non-lipid (pleiotropic) effects of statins in the form of prevention of LDL oxidation, lipid peroxidation and ROS generation mediated by their antioxidant properties are well documented in some of the recent studies^35,36. These pleiotropic effects have been shown to improve the endothelial dysfunction thereby decreasing mortality from cardiovascular deaths independent of lipid lowering effects.

15.Others: Beneficial effects of antioxidant supplementation have also been advocated against detrimental effects of environmental contaminants⁹⁰.

The biggest doubt, which antioxidants raises is that of suicidal oxidative stress, induced by certain antioxidants, like coumarins⁹¹, flavonoids⁹², superoxide dismutase⁹³ and β-carotene⁹⁴. These antioxidants can act as pro-oxidants in certain conditions like presence of transition metals⁹² or at high concentrations⁹¹ and can cause the cell to undergo severe oxidative stress ultimately resulting in suicidal cell death. Recent study⁹⁵ also questions the rationale for antioxidant vitamin supplementation in healthy individuals without any reason. In addition number of questions like appropriate timing of administration, dosage and duration of antioxidant therapy still need to be determined. As one of the study⁹⁶ pointed that different timing of administration, dose and duration of antioxidant vitamins have variable effects on proatherogenic serum markers. Delivery at specific site of free radical generation and to understand the riddle that free radicals are cause or consequence of disease, still remains a challenge ahead in the antioxidant therapy. Many present studies suggest that dietary factors based on cereals, pulses, spices, dark green leafy vegetables such as kale and spinach, citrus fruits, crude palm oil, soybean oil, cod liver oil, sprouts, peppers, whole grain, honey, walnuts and black tea can significantly increase the hepatic antioxidant enzymes and their supplementation reduces the risk of coronary heart disease, cancers and other pathological conditions effectively and safely^{9,11,29,30,63,97,98}. Therefore, due to some of the concerns associated with the use of pharmacological/synthetic antioxidants and the fact that dietary antioxidants supplementation is as effective and safe, it is recommended that all good sources of natural antioxidants should be increased in the diet for prevention and treatment of various pathological conditions.

CONCLUSION:

Free radical/reactive oxygen species generation is a continuous process in the biological system and there are substantial evidences for their involvement in many pathophysiological states where antioxidants can play very important role. But there are number of questions at present associated with the practical application of antioxidant therapy like appropriate timing of administration, choice of antioxidants, dose and duration of therapy to be employed, delivery at specific site & specificity of targeting free radicals, which still need to be determined. Presently, there are convincing evidences suggesting increase intake of antioxidants to be protective in many pathological conditions. However, there are conflicting studies also which not only suggest antioxidant supplementation to be ineffective but in certain conditions detrimental to health also. Moreover, irrational and non-judicial use of antioxidants can also increase the risk of potential toxicity as many antioxidants can also act as pro oxidants under a range of circumstances inspite these concerns they have gained very important status presently. However, under the shadow of these concerns associated with the use of synthetic/pharmacological antioxidants, the best recommended action is to increase the intake of natural dietary antioxidant vitamins by consuming cereals, pulses, nuts, fruits, vegetables, which seems to be a safe and effective approach currently.

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Dr Vishal Tandon received doctorate in Pharmacology from Nagpur University, Maharashtra (India). He authored and/or coauthored over 40 publications in many national and international journals. His research interests include herbal drug screening, especially working on plant vitex negundo. He is currently working as Senior demonstrator, Post graduate department of pharmacology and therapeutics, GMC, Jammu (J&K)-India. His current job responsibilities include teaching UG/PG classes as well as supervising research.

Currently he is Editorial Secretary for JK-SCIENCE, Journal of Medical Education &Research. This journal is indexed in Excerpta Medica/EMBASE, Ulrich periodical Dictionary& Indian Science Abstract and also he is Secretary to Indian Rheumatology Association-J&K CHAPTER. He is life member of Indian Pharmacological society , Association of Physiologist and Pharmacologist of India and Indian Rheumatology Association .

Corresponding address:

Dr Vishal. R. Tandon (MD) (Senior Demonstrator) , Post Graduate Department of Pharmacology & Therapeutics GMC, Jammu (J&K) India - 180001. E-mail: dr_vishaltandon@yahoo.com Phone: 9419195126.