Various ROS are formed continuously as by-products of aerobic metabolism, through reactions with drugs and
environmental toxins. When their level is increased or the level of antioxidants is diminished, it creates a condition called oxidative stress. The ROS can cause serious chemi- cal damage to deoxyribonucleic acid (DNA), proteins, car- bohydrates and unsaturated lipids and can eventually lead to cell death. The ROS have also been implicated in a num- ber of pathological processes including ischemia, cancer, diabetes, inflammatory disease, etc.
Generation of Free Radicals
1. The free radicals are constantly produced during the normal oxidation of foodstuffs, due to leaks in the electron transport chain in mitochondria.
2. Some enzymes such as xanthine oxidase and alde- hyde oxidase form superoxide anion radical or hydro- gen peroxide.
3. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the inflammatory cells (neu- trophils, eosinophils, monocytes and macrophages) produces superoxide anion by a process of respiratory burst during phagocytosis. The superoxide is convert- ed to hydrogen peroxide and then to hypochlorous acid (HCIO) with the help of superoxide dismutase (SOD) and myeloperoxidase (MPO). Along with the activa- tion of macrophages, the consumption of oxygen by the cell is increased drastically; this is called respira- tory burst. In chronic granulomatous disease (CGD),
Fig. 10.1: Formation of reactive oxygen species from molecular
Chapter 10: Xenobiotics 119
the NADPH oxidase is absent in macrophages and neutrophils.
4. Macrophages also produce NO from arginine by the enzyme nitric oxide synthase.
5. Light of appropriate wavelengths can cause photolysis of oxygen to produce singlet oxygen.
6. Peroxidation is catalyzed by lipoxygenase in platelets and leukocytes.
LIPID PEROXIDATION
Free radical-induced peroxidation of membrane lipids oc- curs in three stages—initiation, propagation and termination.
Initiation Phase
Initiation step involves the removal of hydrogen atom (H) from polyunsaturated fatty acids (RH), caused by hydroxyl radical.
RH + OH• R• + H2O
Propagation Phase
Under aerobic conditions, the fatty acid radical (R•) takes up oxygen to form peroxyl radical (ROO•). The latter, in turn, can remove H– atom from another polyunsaturated fatty
acid (PUFA) (RH) to form lipid hydroperoxide (ROOH).
R• + O
2 ROO •
ROO• + RH ROOH + R•
The hydroperoxides are capable of further stimulating lipid peroxidation, as they can form alkoxy (RO•) and per-
oxyl (ROO•) radicals.
Termination Phase
Lipid peroxidation proceeds as a chain reaction until the available PUFA gets oxidized (Fig. 10.2).
Role of Free Radicals in Diseases
They have been found to play significant role in diseases ranging from the diabetes mellitus to arthritis to acquired immunodeficiency syndrome (AIDS) and infertility: 1. Lipid peroxidation and oxidative process are said to be
one of the important factors contributing to the patho- genesis of atherosclerosis.
Fig. 10.2: Polyunsaturated fatty acid
2. Many substances including free radicals induce ge- nomic damage that play important role in number of neoplastic diseases.
3. Diabetes has also been found to be associated with increased free radical damage. In diabetic patients,
serum thiobarbituric acid reactive material (a well-
known biomarker for assessing free radical mediated- tissue damage) is found to be elevated.
4. Increased lipid oxidation and oxidative stress has been attributed to the increased plasma glucose level. 5. Free radicals have been found to be associated with
the aging phenomenon.
6. Accumulation of lipofuscin (the so-called age pig-
ment) has been correlated with the aging.
7. Inflammatory diseases: Rheumatoid arthritis is a
chronic inflammatory disease. The free radicals pro- duced by neutrophils are the predominant causative agents.
8. Respiratory diseases: Direct exposure of lungs to
100% oxygen for a long period (more than 24 hours) is known to destroy endothelium and cause lung edema. This is mediated by free radicals. ROS are also respon- sible for adult respiratory distress syndrome (ARDS), a disorder characterized by pulmonary edema.
9. Cataract: Increased exposure to oxidative stress con-
tributes to cataract formation.
10. Retrolental fibroplasia seen in premature infants is
due to free radicals.
11. Skin diseases such as psoriasis and leukoderma may
result.
Free Radical Scavenger Systems
Superoxide Dismutase
The mitochondrial SOD is manganese dependent; cyto- plasmic enzyme is copper-zinc dependent. SOD is a non- heme protein.
Clinical correlation: A defect in SOD gene is seen in patients
with amyotrophic lateral sclerosis (Lou Gehrig’s disease).
SOD O2+ O2 + 2H+ H 2O2 + O2 Catalase 2H2O2 2H2O + O2 Glutathione Peroxidase
In the next step, the H2O2 is removed by glutathioneperoxi- dase (POD). It is a self-dependent enzyme.
Glutathione Reductase
The oxidized glutathione, in turn, is reduced by glutathi- one reductase (GR), in presence of NADPH. This NADPH is generated with the help of glucose-6-phosphate dehy- drogenase (GPD) in hexose monophosphate (HMP) shunt pathway.
Catalase
When H2O2 is generated in large quantities the enzyme catalase is also used for its removal.
Catalase
2 H2O2 O2 + 2H2O
Polyphenols
Consumption of polyphenol-rich fruits, vegetables and beverages is beneficial to human health.
Dietary polyphenols represent a wide variety of com- pounds that occur in fruits, vegetables, wine, tea and chocolate. They contain flavonoids, isoflavones, flavonols, catechins and phenolic acids.
They shows antioxidant, antiapoptosis, antiaging, an- ticarcinogenic, anti-inflammatory, antiatherosclerotic ef- fects. They are protective against cardiovascular diseases. Grape polyphenols can prevent brain damage due to al- cohol. Oral administration of grape polyphenol extract ameliorates cerebral ischemia-induced neuronal carnage. Grape-seed procyanidins prevent lower grade inflamma- tion by modulating cytokine expression in rats.
ANTIOXIDANTS
A biological antioxidant may be defined as a substance (present in low-concentrations compared to an oxidizable substrate) that significantly delays or inhibits oxidation of a substrate. Antioxidants may be considered as the scaven- gers of free radicals.
Role of Antioxidants
Apart from the scavenging enzymes, there are two types of antioxidants.
Preventive Antioxidants
The antioxidants will inhibit the initial production of free radicals. They are catalase, glutathione peroxidase and eth- ylenediaminetetraacetate (EDTA).
Chain-breaking Antioxidants
The antioxidants can inhibit propagative phase. They in- clude superoxide dismutase, uric acid and vitamin E. The
a-tocopherol (T-OH) (vitamin E) would intercept the peroxyl free radical and inactivate it before a PUFA can be attacked.
T-OH + ROO TO’ + ROOH
TO’ + ROO inactive products
Vitamin E (a-tocopherol) acts as the most effective naturally occurring chain-breaking antioxidant in tissues.
Important Antioxidants
• Vitamin E is the lipid phase antioxidant • Vitamin C is the aqueous phase antioxidant
• Ceruloplasmin can act as an antioxidant in extracellu-
lar fluid
• Caffeine is an effective antioxidant
• Cysteine, glutathione and vitamin A are minor antioxi-
dants. Beta carotene can act as a chain-breaking anti-
oxidant, but is less effective than a-tocopherol.
Antioxidants Used as Therapeutic Agents
1. Vitamin E. 2. Vitamin C. 3. Dimethylthiourea. 4. Dimethyl sulfoxide. 5. Allopurinol.
Commercial Use of Antioxidants
Antioxidants are regularly used in food industry to in- crease the shelf-life of products. Commercially used food preservatives are vitamin E, propyl gallate, butylated hy- droxyanisole (BHA) and butylated hydroxytoluene. They prevent oxidative damage of oils, particularly those con- taining PUFA and prevent rancidity.