**Use of CoA Biosynthesis Modulators and Selenoprotein Model Substance in Correction of Brain Ischemic and Reperfusion Injuries**

Nina P. Kanunnikova, Natalya Z. Bashun and Andrey G. Moiseenok

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/46077

## **1. Introduction**

492 Lipid Peroxidation

Waris G, Huh KW & Siddiqui A . (2001). Mitochondrially associated hepatitis B virus X protein constitutively activates transcription factors STAT-3 and NF-kappa B via

oxidative stress. Mol Cell Biol 21:7721-7730.

Acute disruption in brain blood circulation is a widespread cause of death and the most frequent cause of health loss in most countries of the world. About 6 millions of people suffer from stroke every year and this number is constantly increasing. Stroke has a high mortality rate – up to 30% of patients die. Only about 20% of surviving patients manage to return to their previous occupation. Most of patients are unable to take care of themselves and need help of relatives or medical personnel [1, 2]. About 80-85% of all cases of stroke are ischemic strokes. Therefore protecting brain from ischemia-induced damage is in the focus of modern neuropathology and neurosurgery studies, especially due to the increase in the number of neurosurgical operations which might cause additional blood flow impairements.

The severity of injuries of physiological reactions and biochemical processes caused by blood flow impairements depends on the degree of blood flow disruption in brain (fig.1) [1, 3].


**Figure 1.** Correspondence between brain tissue changes and blood flow disruption

© 2012 Kanunnikova et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Decline of partial pressure of oxygen, significant decrease of ATP and glucose levels, membrane depolarization, extremely high levels of extracellular glutamate and intracellular calcium ions - all these factors contribute to development of the aforementioned injuries in the nervous tissue [4, 5, 6]. For example, higher level of calcium ions leads to stimulation of phospholipases and proteases, and activation of glutamate NMDA-receptors which in turn increase activity of nNoS and eNOS isoforms. As the result, amplification of lipoperoxidation takes place.

Disruption of electron transfer and oxidative phosphorilation within mitochondria are the first manifestations of ischemia-induced damages in the brain [7, 8, 9, 10], and the basic object of the injuries are presumably mitochondrial membranes [11, 12, 13, 14]. On this basis, ability of the brain to restore its functions following ischemia and reperfusion depends mainly on three processes - depletion of energic resources, excessive accumulation of excitatory amino acids [15, 16, 17], and formation of reactive oxygen species caused by leakage of electrons from intermediate links of respiratory change [18, 19, 20].

#### **2. Features of oxidative stress in brain tissue during ischemia-reperfusion**

Brain ischemia causes formation of free radical forms of oxygen which induce damage of neuronal membranes and biomacromolecules, particularly nucleic acids and proteins. Brain tissue has heightened disposition to development of oxidative stress. Brain cell membranes have high concentrations of polyunsaturated fatty acids which are the main substrate of free radical reactions [21, 22, 23]. When a free radical appears in membrane chance for its interaction with fatty acid is increased as a number of unsaturated links is rised. Unsaturated fatty acids provide more fluidity for membranes, therefore their changes caused by more active lipoperoxidation lead to increase of their viscosity and injuries of their barrier functions. It is known that synapse plasmatic membranes contain higher level of polyunsaturated fatty acids than myelin membranes. Many functionally important neuronal proteins are membranebound and depend on lipid environment. Simultaneously, system of antioxidant protection in brain has obviously less capacity than in other tissues, and enzymatic components of the system in brain are more sensitive to oxidative action [24, 25].

The second danger of lipoperoxidative activation in brain lies in the fact that disruption of nerve membrane integrity leads to increasing release of "excitotoxic" transmitters, such as glutamate, aspartate, etc [15, 16, 26-28].High rate of biogenic amine metabolism in brain leads to formation of ROS [29]. For example, monoamine oxidase reaction is linked with H2O2 formation. This phenomenon may be an additional source for generation of active radical products which are able to initiate lipoperoxidation in the presence of metals with variable valence. Dopamine, its precursor L-DOPA, 5-hydroxytraptamine, and norepinephrine may generate О2∙ not only, but quinones/semiquinones, too, which may decrease GSH level and bound with protein SH-groups. Oxidation can be catalized by transitional metal ions. Maximal increase for free radical generation and following activation for lipoperoxidation takes place in postischemic time – during recovery of blood circulation in brain tissue [30-33]. Nevertheless, possibility for formation of free radicals at earlier stage of brain damage exists during ischemia, too [34]. Enhancement of redox state of mitochondrial respiratory chain in these conditions gives an opportunity for oxygen to interact with intermediate components of the respiratory chain, for example ubisemiquinone [7]. This process takes the path of one-electron reduction of molecular oxygen and leads to formation of superoxide-anion. Consequently, heightened formation of free radicals may take place in tissues with insufficient blood circulation and decreased partial oxygen pressure [35]. Studies in mice and rats with genetic deficiency of superoxide dismutase confirmed the important role of free radicals in neuronal death/survival during brain ischemia [36, 37].In the postischemic period (during recirculation) when oxygen actively absorbs by brain tissue, oxygen radicals generation is caused by activation of enzymatic processes, too (arachidonic acid cascade, xantine oxydase system, activation of NADPHoxydase in polymorphonuclear leucocytes) [38-40].

494 Lipid Peroxidation

lipoperoxidation takes place.

Decline of partial pressure of oxygen, significant decrease of ATP and glucose levels, membrane depolarization, extremely high levels of extracellular glutamate and intracellular calcium ions - all these factors contribute to development of the aforementioned injuries in the nervous tissue [4, 5, 6]. For example, higher level of calcium ions leads to stimulation of phospholipases and proteases, and activation of glutamate NMDA-receptors which in turn increase activity of nNoS and eNOS isoforms. As the result, amplification of

Disruption of electron transfer and oxidative phosphorilation within mitochondria are the first manifestations of ischemia-induced damages in the brain [7, 8, 9, 10], and the basic object of the injuries are presumably mitochondrial membranes [11, 12, 13, 14]. On this basis, ability of the brain to restore its functions following ischemia and reperfusion depends mainly on three processes - depletion of energic resources, excessive accumulation of excitatory amino acids [15, 16, 17], and formation of reactive oxygen species caused by

**2. Features of oxidative stress in brain tissue during ischemia-reperfusion** 

Brain ischemia causes formation of free radical forms of oxygen which induce damage of neuronal membranes and biomacromolecules, particularly nucleic acids and proteins. Brain tissue has heightened disposition to development of oxidative stress. Brain cell membranes have high concentrations of polyunsaturated fatty acids which are the main substrate of free radical reactions [21, 22, 23]. When a free radical appears in membrane chance for its interaction with fatty acid is increased as a number of unsaturated links is rised. Unsaturated fatty acids provide more fluidity for membranes, therefore their changes caused by more active lipoperoxidation lead to increase of their viscosity and injuries of their barrier functions. It is known that synapse plasmatic membranes contain higher level of polyunsaturated fatty acids than myelin membranes. Many functionally important neuronal proteins are membranebound and depend on lipid environment. Simultaneously, system of antioxidant protection in brain has obviously less capacity than in other tissues, and enzymatic components of the

The second danger of lipoperoxidative activation in brain lies in the fact that disruption of nerve membrane integrity leads to increasing release of "excitotoxic" transmitters, such as glutamate, aspartate, etc [15, 16, 26-28].High rate of biogenic amine metabolism in brain leads to formation of ROS [29]. For example, monoamine oxidase reaction is linked with H2O2 formation. This phenomenon may be an additional source for generation of active radical products which are able to initiate lipoperoxidation in the presence of metals with variable valence. Dopamine, its precursor L-DOPA, 5-hydroxytraptamine, and

decrease GSH level and bound with protein SH-groups. Oxidation can be catalized by transitional metal ions. Maximal increase for free radical generation and following activation for lipoperoxidation takes place in postischemic time – during recovery of blood circulation in brain tissue [30-33]. Nevertheless, possibility for formation of free radicals at

not only, but quinones/semiquinones, too, which may

leakage of electrons from intermediate links of respiratory change [18, 19, 20].

system in brain are more sensitive to oxidative action [24, 25].

norepinephrine may generate О2∙-

The fact that ischemia itself is unable to increase level of lipoperoxidation intermediate products is not surprising because during hypoxia amount of molecular oxygen is insufficient for observable activation of lipoperoxidation in brain tissue. Nevertheless, in these conditions the amount of hydroxyperoxides is increased and lag period for lipoperoxidation activation becomes shorter, which serves as evidence for decrease of antioxidant protection and increased formation of superoxide oxygen anion in brain tissue [41]. Herewith level of endogenic antioxidants in brain may be unchanged [42, 43]. For example, α-tocoferol level in rat brain is unchanged following 80 min after occlusion of middle cerebral artery and subsequent reperfusion. Unchanged levels of antioxidants were observed following bilateral occlusion of arteria carotis and reperfusion in gerbils, too [44, 45].

Recently a concept on polyfunctional physiologic role of free radicals in organism and in brain especially, is declared [42, 43, 46-48]. On the one hand, they act on key cell enzymes and receptors inside cells and cause destructive processes in tissues. On the other hand, they play a role of second messengers and may help with cell adaptive reactions to changed environmental conditions. Therefore low efficiency of antioxidant therapy by substances which bound free radical excess during ischemic injury treatment is not surprising [49, 50, 51]. In addition, these drugs have low bioavailability and must be used for at least several weeks before any effect can be observed [52, 53, 54-56].

It is necessary to emphasize that the main defense from excessive amounts of free radicals formed within cells is the action of antioxidant protection enzymes, such as selenoproteins, but not the action of low molecular wieght antioxidants. Under normal conditions these enzymes are sufficient for maintenance of low safe levels of free radicals, but in reperfusional conditions their activity is insufficient for maintenance pro- and antioxidant balance. Earlier attempts to administer substances of superoxide dismutase and catalase enzymes to animals in experimental models were unsuccessful because they poorly penetrate blood-brain barrier and cell membranes [51, 53].

Further research of neuroprotection in this direction is not very promising because activation of lipoperoxidation in phospholipid structures of nerve cell membranes is

eliminated by the system of superoxide dismutase – catalase to a small degree [53]. Detoxication in these structures is primarily carried by enzymes of glutathione cycle, selenium-cystein-comprising glutathione peroxidases [49, 54, 55, 57-59].

## **3. Role of glutathione in mechanisms of antioxidant protection in brain**

Glutathione cycle is the most important antioxidant system in brain cells [59-66]. Glutathione protects cells against reactive oxygen, nitrogen and other species. As an antioxidant it is involved in the detoxication of malonic dialdehyde, 4-hydroxy-2-nonenal and other products of lipoperoxidation. The glutathione couple GSH/GSSG takes part in maintaining cellular redox status [67, 68]. Glutathione is presumably a key participant of the defense system in brain cell [69-71].

Increased level of oxidized form of glutathione and changes of glutathione system activity occur at early stages of oxidative stress and may be marks of the severity of oxidative stress [71-75]. Hydroxyl radical and nitric oxide or peroxynitrite may interact directly with GSH leading to GSSG formation. Hydrogen peroxide may be removed by catalase or by glutathione peroxidase [76].

GSH is present in cytoplasma, endoplasmatic reticulum, nucleus, and mitochondria. In most of the compartments GSH is found predominantly in highly reduced state (about 99% of the total level of glutathione). Glutathione peroxidase is localized mainly in cytosol, too, whereas catalase is found mostly in peroxisomes. The affinity of glutathione peroxidase for H2O2 is one to two orders of magnitude higher than that of catalase, and catalase is less active in brain than in other tissues.

GSH is synthesized from cysteine, glutamate and glycine. Neurones have lower GSH level and use a more limited list of substrates for GSH synthesis, but they use glutamine for GSH synthesis more effectively than astrocytes because they have glutaminase for formation of glutamate from glutamine. Neurones can not absorb cystine but they actively carry off cysteine [65], so availability of cysteine influences at the GSH level inside neurones. At the same time maximal rate for GSH synthesis within astrocytes is observed in the presence of glutamate, cystine and glycine.

GSSG restores by glutathione reductase into GSH in the presence of NADPH (salvage cycle), which originated mainly from pentose phosphate pathway of metabolism. About 3-5% of oxygen in brain is consumed this way.

In physiological conditions GSSG level both in neurones and astrocytes reach no more than 1% of total content of glutathione in tissue but during oxidative stress it may be about 40% of total value of the glutathione in the astrocyte culture. One astrocyte cell may effectively protect 20 neurons from peroxides but lack of glucose greatly decreases capacity of astrocytes to bind peroxides. It has been shown that pentose phosphate pathway in astrocyte culture is very sensitive to peroxide action.

Decrease of total glutathione content and decline of GSH/GSSG ratio are indicators of the severity of oxidative stress in ischemic brain tissue [67]. It is known that decrease of GSH level leads to aggravation of ischemia-induced injuries, while increase of its level leads to opposite result. Glutamate may facilitate decrease of GSH level because it inhibits use of cysteine which is required for glutathione synthesis by cells [67]. Genetic failure of a cell glutathione peroxidase makes rats more susceptible to neurotoxins and brain ischemia [68]. Excessive glutathione peroxidase expression in transgenic mice leads to prevention of irreversible hypoxia-induced changes. Decline of GSH concentration may weaken the stability of an organism to hypoxia both by inactivation of pentose phosphate way enzymes as by inhibition of thioenzymes of tissue respiration chain [76, 77]. These disruptions cause development of energy deficiency which is the main chain of biochemical mechanism of tissue hypoxia. In addition thiol-disulfide metabolism changes may form the basis for mechanisms of disconnection of the oxidation and phosphorilation processes [78-80]. As a result, use of oxygen in biological oxidation processes may be broken and become a base for pathogenetic component of intiation and generalization of oxidative stress.

496 Lipid Peroxidation

defense system in brain cell [69-71].

active in brain than in other tissues.

glutamate, cystine and glycine.

oxygen in brain is consumed this way.

astrocyte culture is very sensitive to peroxide action.

glutathione peroxidase [76].

eliminated by the system of superoxide dismutase – catalase to a small degree [53]. Detoxication in these structures is primarily carried by enzymes of glutathione cycle,

**3. Role of glutathione in mechanisms of antioxidant protection in brain** 

Glutathione cycle is the most important antioxidant system in brain cells [59-66]. Glutathione protects cells against reactive oxygen, nitrogen and other species. As an antioxidant it is involved in the detoxication of malonic dialdehyde, 4-hydroxy-2-nonenal and other products of lipoperoxidation. The glutathione couple GSH/GSSG takes part in maintaining cellular redox status [67, 68]. Glutathione is presumably a key participant of the

Increased level of oxidized form of glutathione and changes of glutathione system activity occur at early stages of oxidative stress and may be marks of the severity of oxidative stress [71-75]. Hydroxyl radical and nitric oxide or peroxynitrite may interact directly with GSH leading to GSSG formation. Hydrogen peroxide may be removed by catalase or by

GSH is present in cytoplasma, endoplasmatic reticulum, nucleus, and mitochondria. In most of the compartments GSH is found predominantly in highly reduced state (about 99% of the total level of glutathione). Glutathione peroxidase is localized mainly in cytosol, too, whereas catalase is found mostly in peroxisomes. The affinity of glutathione peroxidase for H2O2 is one to two orders of magnitude higher than that of catalase, and catalase is less

GSH is synthesized from cysteine, glutamate and glycine. Neurones have lower GSH level and use a more limited list of substrates for GSH synthesis, but they use glutamine for GSH synthesis more effectively than astrocytes because they have glutaminase for formation of glutamate from glutamine. Neurones can not absorb cystine but they actively carry off cysteine [65], so availability of cysteine influences at the GSH level inside neurones. At the same time maximal rate for GSH synthesis within astrocytes is observed in the presence of

GSSG restores by glutathione reductase into GSH in the presence of NADPH (salvage cycle), which originated mainly from pentose phosphate pathway of metabolism. About 3-5% of

In physiological conditions GSSG level both in neurones and astrocytes reach no more than 1% of total content of glutathione in tissue but during oxidative stress it may be about 40% of total value of the glutathione in the astrocyte culture. One astrocyte cell may effectively protect 20 neurons from peroxides but lack of glucose greatly decreases capacity of astrocytes to bind peroxides. It has been shown that pentose phosphate pathway in

selenium-cystein-comprising glutathione peroxidases [49, 54, 55, 57-59].

## **4. Role of energy metabolism changes in mechanisms of brain tissue ischemia-induced injuries**

Brain is very sensitive to disruptions of energy metabolism processes beacause brain tissue requires constant supply of energy substrates whereas sources for energy formation in brain are rather limited, turnover of metabolism is high, and metabolism is dependent on aerobic oxidation of glucose and constant supply of oxygen in a great extent [81, 82] Maintenance for electric neuron activity and rate of impulse passage depend directly on presence and availability of energy substrates, too [83, 84].

Brain tissue cannot metabolize fatty acids therefore the main source for energy formation in brain is glucose. Nevertheless, during focal brain ischemia, increase of glucose level does not help cells to prevent ischemic injuries and also promotes their structural and functional damage [85]. Mechanisms of these changes include shift of pH to an acid side inside cell, increase of permeability of blood-brain barrier, infiltration of brain parenchyma by neutrophiles, accumulation of extracellular glutamate, and unfavorable corticosterone action. Intensive metabolism of glucose in the penumbra region may promote increasing acidic reaction of the medium, promote attraction of neutrophiles in the region.

Limitation of metabolic consumption of glucose in ischemic brain tissue may have protective effect, especially in such conditions when its metabolism will be faster or other source of fuel will be used. Possibilities for replacement of glucose in brain tissue are rather limited [86-89]. Lactic acid may be an alternative source for energy formation in brain in certain conditions because the glucose is metabolized presumably in glial cells whereas in neurones energy metabolism is based presumably on lactate oxidation [90, 91]. There is some evidence that the process is particularly important for maintenance of vital functions during postischemic time. For example, decrease of lactate transfer through plasmatic mebrane in brain following preliminary whole ischemia causes neurone injuries. From clinic

practice it is known that consumption of lactate or pyruvate during brain ischemia show neuroprotective actions of the substances [90, 91]. Presence of adequate concentrations of pyruvate facilitate for maintenance of stable level of membrane potentials and proton gradient on vesicular membranes [87, 92].

Brain ischemia is different from other types of ischemia because oxygen deficiency causes significant changes in the oxidating process of energy substrates which are present in brain in suffucient quantities [20, 42]. Anaerobic glycolisis as alternative way for energy supply is not substantial for supporting ATP stock in nerve tissue during compensated and decompensated stages of hypoxia [11].

Aerobic energy formation is the basic process for nerve tissue, but starts to fail before oxygen concentration falls below critical level, because hypoxia influences kinetic properties of respiratory chain enzymes. During early stages of ischemia energy functions of mitochondria already start changing: conjugacy of oxidative phosphorilation process and regulatory control by ATP becomes weaker, rate of inphosphorilated respiration increases. Shift of ratio NAD/NADH occurs to the side of NADH, as a result final stages of the Krebs' cycle are inhibited, and activation of succinate oxidase stage takes place. This way allows to maintain oxidative phosphorilation and respectively macroergic substance production at sufficient level for some time. "Oxygen hunger" already at early stages of hypoxia leads to beginning of relative "substrate hunger" - energy substrates are not being oxydated while they are still available. This is a characteristic property for ischemia [93].

There are only 2 ways of restoration of brain metabolism after stroke and hypoxia – restoration of NAD-dependent part of the Krebs' cycle and stimulation of alternative path of metabolism, succinate oxidation.

Succinic acid is an intermediate of the cytric cycle which supports formation of macroergic phosphates and reductive equivalents in the conditions with physical loadings and stress [94-97]. Oxidation of succinic acid is the most potent energy process inside mitochondria, and during stress this process becomes even more important due to succinate dehydrogenase activation. Depletion of endogenic succinic acid may be a reason explaining inability of tissue to maintain reaction of activation of energy processes for a long time [98]. If NADH and СО2 are present in excess, conversion of reactions of second part of the cytrate cycle in which NADH is consumed takes place - from oxaloacetate to succinate, and that fact supports reactions of the first part of the cytrate cycle reactions which require oxidized NAD and promote for additional accumulation of succinate.

Another important result of the bioenergic hypoxia is damage of ion pump action and ion imbalance in the form of excessive accumulation of intracellular calcium, sodium, chlorine [99]. The intracellular calcium excess leads to activation of phospholipase A, damage of cell membranes and release of arachidonic acid take place. As a result, lipoperoxidation activates and causes following cell membrane damages, neuron depolarization and release of excitotoxic amino acids, especially glutamate, in extracellular space [99].

Thereby adaptive effects of succinate derivatives are related to their property to induce compensatory metabolic flows in mitochondrial respiratory chain ("succinate oxidase" way) in extreme conditions, to provide replenishment for cytoplasmatic pool by reduced forms of NAD and NADP, to accelerate ATP formation, change over energy formation from NADdependent to FAD-dependent way, eliminate an excess of acetyl-CoA, support activity of the Krebs' cycle in hypoxic conditions, stabilize membrane potential of mitochondria and cell membranes. Advantage of succinate oxidase way versus NAD-dependent substrates in competition for respiratory chain is amplified in the hypoxia conditions because flavines (flavoproteins) continue in oxygenated form longer than pyridine nucleotides.

498 Lipid Peroxidation

gradient on vesicular membranes [87, 92].

decompensated stages of hypoxia [11].

metabolism, succinate oxidation.

and promote for additional accumulation of succinate.

practice it is known that consumption of lactate or pyruvate during brain ischemia show neuroprotective actions of the substances [90, 91]. Presence of adequate concentrations of pyruvate facilitate for maintenance of stable level of membrane potentials and proton

Brain ischemia is different from other types of ischemia because oxygen deficiency causes significant changes in the oxidating process of energy substrates which are present in brain in suffucient quantities [20, 42]. Anaerobic glycolisis as alternative way for energy supply is not substantial for supporting ATP stock in nerve tissue during compensated and

Aerobic energy formation is the basic process for nerve tissue, but starts to fail before oxygen concentration falls below critical level, because hypoxia influences kinetic properties of respiratory chain enzymes. During early stages of ischemia energy functions of mitochondria already start changing: conjugacy of oxidative phosphorilation process and regulatory control by ATP becomes weaker, rate of inphosphorilated respiration increases. Shift of ratio NAD/NADH occurs to the side of NADH, as a result final stages of the Krebs' cycle are inhibited, and activation of succinate oxidase stage takes place. This way allows to maintain oxidative phosphorilation and respectively macroergic substance production at sufficient level for some time. "Oxygen hunger" already at early stages of hypoxia leads to beginning of relative "substrate hunger" - energy substrates are not being oxydated while

There are only 2 ways of restoration of brain metabolism after stroke and hypoxia – restoration of NAD-dependent part of the Krebs' cycle and stimulation of alternative path of

Succinic acid is an intermediate of the cytric cycle which supports formation of macroergic phosphates and reductive equivalents in the conditions with physical loadings and stress [94-97]. Oxidation of succinic acid is the most potent energy process inside mitochondria, and during stress this process becomes even more important due to succinate dehydrogenase activation. Depletion of endogenic succinic acid may be a reason explaining inability of tissue to maintain reaction of activation of energy processes for a long time [98]. If NADH and СО2 are present in excess, conversion of reactions of second part of the cytrate cycle in which NADH is consumed takes place - from oxaloacetate to succinate, and that fact supports reactions of the first part of the cytrate cycle reactions which require oxidized NAD

Another important result of the bioenergic hypoxia is damage of ion pump action and ion imbalance in the form of excessive accumulation of intracellular calcium, sodium, chlorine [99]. The intracellular calcium excess leads to activation of phospholipase A, damage of cell membranes and release of arachidonic acid take place. As a result, lipoperoxidation activates and causes following cell membrane damages, neuron depolarization and release

of excitotoxic amino acids, especially glutamate, in extracellular space [99].

they are still available. This is a characteristic property for ischemia [93].

Disruption of energy metabolism can be mainly observed at the stage of succinate formation. That may be caused by oxidative stress-induced changes of stable state of plasmatic and mitochondrial membranes and changes in activity of membrane-bound enzymes of the Krebs cycle and GABA bypass [100, 101]. Significant activation of the GABA bypass enzymes takes place during ischemia-reperfusion which not only causes raise of succinate formation, but also leads to increasing formation of gamma-hydroxybutyric acid through reductase reaction. GHBA has protective effect against changes of energy formation processes in brain tissue during hypoxia [102]. As activity of glutamate dehydrogenase in brain is rather low compared with other tissues, GABA bypass plays a key role in compensatory maintenance of succinate level sufficient for adequate metabolism in "succinate oxidase" way in different extremal situations, for example in brain ischemiareperfusion conditions.

Succinic acid derivatives are effective modern antioxidants in the brain because succinate regulates activity of SDH in the Krebs' cycle and restores activity of respiratory mitochondrial chain not only, but increases microcirculation in tissues.

## **5. Role of CoA in mechanisms of neuroprotection in brain ischemiareperfusion conditions**

Beneficial effects of precursors of CoA biosynthesis, such as pantothenic acid and its derivatives, include protection from lipoperoxidation and supporting membrane structure, and these effects have been observed in radiation injury, miocard ischemia, diabetes mellitus, CCl4 -intoxication, heavy hypothermia, etc [103-106]. Protective action of pantothenate derivatives have been reported in situations accompanied by oxidative stress, for example, in experimental ischemia-reperfusion of myocardium. It has been shown that antioxidative and membrane-protective effects of the pantothenate derivatives are accompanied by an activation of biosynthesis CoA system and increasing of intracellular level of a free CoA [108, 109]. Presumably, the mechanism of cell protection is CoAdependent or realized through CoA-(acyl-CoA)-dependent biochemical reactions, including rise of intracellular glutathione level and maintenance of its redox status.

It is believed that the physiological function of CoA system is participation in formation of redox potential of glutathione and proteins, redox signaling and maintenance of biological membrane stability, especially in brain tissue [104, 105, 106].

The CoA biosynthesis system is a group of very stable continuously active self-regulated processes focused on maintaining stability of intramitochondrial CoA-SH (up 70-80% of the total cell value). This function maintains constant flow of oxidative substrates and their effective using for ATP formation in the citric acid cycle [109, 110, 111].

The lesser CoA pool in cytosol where acyl-CoA is used in biosynthetic processes (biosynthesis of phospholipids, fatty acids) is studied to a lesser extent. "Turnover" pool of the coenzyme takes part in reactions of carnitine-dependent transfer of fatty acid residues and acetate through mitochondrial membranes [105]. The main events for interrelations between specifically bound cytosolic CoA-S-S-protein, CoA-S-S-glutathione, free and proteidized glutathione take place within cytosolic compartment (including endoplasmatic reticulum) presumably due to thiol-disulfide-exchange reactions which provide stability during limited variations of redox potential and support a realization of redox sygnaling. Based on this hypothesis, the capability of CoA biosynthesis precursors in low concentrations (0.1-1 мМ) or in vivo experiments prevents lipoperoxidation activation, damages of membrane integrity initiated by different physical or chemical factors. The obligatory condition of the above-mentioned defensive effect is biotransformation of pantothenate derivatives into CoA and significant increase of intracellular GSH level. The process is highly specific because homopantothenic acid which is similar to pantothenic one in terms physical and chemical properties can not transform into CoA, does not increase intracellular glutathione level and does not protect plasmatic membrane stability in cell culture [112].

Additional effects of the CoA precursors in defense of lipoperoxidative activation have also been observed. These include rapid initiation of lipid biosynthesis from labeled precursors, positive influence on mitochondrial energy parameters, as well as protection against apoptosis activation caused by free radical oxidation products [113, 114]. Redox sygnaling process controls the initiation and direction of these processes. The redox potential is determined by the glutathione system predominantly [105, 115]. This data may confirm that the CoA biosynthesis system is the most important factor of intracellular stability of GSH level [103, 105].

Maintenance of sufficiently high CoA biosynthesis activity has an important role in brain because acetyl-CoA is used not only as the main way for glucose intake into the Krebs cycle but is also a substrate for acetylcholine synthesis. The relationship between ability for CoA biosynthesis and activity of acetylcholine metabolism within cholinergic neurones may be an important factor in modulation of their sensitivity to damaging influences [103].

Among the necessary conditions for successful biosynthesis of acetyl-CoA, are presence of CoA precursors inside mitochondria, and also the presence of carnitine which transfers acetyl radicals into mitochondria. Under oxidative stress conditions when a lot of lipoperoxide products are released from membranes as a result of phospholipase activation, the potential for CoA sequestration increases, which includes appearance of hard to metabolize acyl-CoA derivatives. Under these conditions the role of carnitine increases. СоА and L-carnitine are among the key factors of intramitochondrial metabolism of fatty and organic acids, and relationship between their levels represents an essential mechanism for cytosol-mitochondrial process of acyl residue activation and transfer [110, 116]. Based on the main localization of a total CoA within mitochondrial matrix, while carnitine is located in cytosol, the molar ratio of CoA/carnitine may have significant functional role for decrease of long-chain acyl-CoA in cytosol and their accelerated utilization in a β-oxidation process.

500 Lipid Peroxidation

culture [112].

level [103, 105].

The CoA biosynthesis system is a group of very stable continuously active self-regulated processes focused on maintaining stability of intramitochondrial CoA-SH (up 70-80% of the total cell value). This function maintains constant flow of oxidative substrates and their

The lesser CoA pool in cytosol where acyl-CoA is used in biosynthetic processes (biosynthesis of phospholipids, fatty acids) is studied to a lesser extent. "Turnover" pool of the coenzyme takes part in reactions of carnitine-dependent transfer of fatty acid residues and acetate through mitochondrial membranes [105]. The main events for interrelations between specifically bound cytosolic CoA-S-S-protein, CoA-S-S-glutathione, free and proteidized glutathione take place within cytosolic compartment (including endoplasmatic reticulum) presumably due to thiol-disulfide-exchange reactions which provide stability during limited variations of redox potential and support a realization of redox sygnaling. Based on this hypothesis, the capability of CoA biosynthesis precursors in low concentrations (0.1-1 мМ) or in vivo experiments prevents lipoperoxidation activation, damages of membrane integrity initiated by different physical or chemical factors. The obligatory condition of the above-mentioned defensive effect is biotransformation of pantothenate derivatives into CoA and significant increase of intracellular GSH level. The process is highly specific because homopantothenic acid which is similar to pantothenic one in terms physical and chemical properties can not transform into CoA, does not increase intracellular glutathione level and does not protect plasmatic membrane stability in cell

Additional effects of the CoA precursors in defense of lipoperoxidative activation have also been observed. These include rapid initiation of lipid biosynthesis from labeled precursors, positive influence on mitochondrial energy parameters, as well as protection against apoptosis activation caused by free radical oxidation products [113, 114]. Redox sygnaling process controls the initiation and direction of these processes. The redox potential is determined by the glutathione system predominantly [105, 115]. This data may confirm that the CoA biosynthesis system is the most important factor of intracellular stability of GSH

Maintenance of sufficiently high CoA biosynthesis activity has an important role in brain because acetyl-CoA is used not only as the main way for glucose intake into the Krebs cycle but is also a substrate for acetylcholine synthesis. The relationship between ability for CoA biosynthesis and activity of acetylcholine metabolism within cholinergic neurones may be

Among the necessary conditions for successful biosynthesis of acetyl-CoA, are presence of CoA precursors inside mitochondria, and also the presence of carnitine which transfers acetyl radicals into mitochondria. Under oxidative stress conditions when a lot of lipoperoxide products are released from membranes as a result of phospholipase activation, the potential for CoA sequestration increases, which includes appearance of hard to metabolize acyl-CoA derivatives. Under these conditions the role of carnitine increases. СоА and L-carnitine are among the key factors of intramitochondrial metabolism of fatty and

an important factor in modulation of their sensitivity to damaging influences [103].

effective using for ATP formation in the citric acid cycle [109, 110, 111].

Generation and use of succinyl-CoA in mitochondria have a special role for mitochondrial oxidation regulation during oxidative stress caused by ischemia-reperfusion [107, 108]. Chances for alternative succinyl-CoA biosynthesis increase significantly when CoA biosynthesis processes are activated in cytosol. In view of this, data on the effects of carnitine on the activity of the key enzyme of CoA biosynthesis, namely patothenate kinase, has high significance. It has been shown that L-carnitine cancels out inhibitory action of physiological concentrations of dephospho-CoA, CoA-SH, and acetyl-CoA on pantothenate kinase. This enables directed regulation of CoA-dependent metabolic processes following simultaneous injection of carnitine substances and pantothenate derivatives - precursors of CoA biosynthesis - namely, panthenol [117, 118].

Study of changes of CoA level during ischemia or ischemia-reperfusion showed markedly stable ratio and levels of free CoA, short-chain acyl-CoA, and on the whole the acid-soluble fraction of CoA, in hemispheres during ischemic damage [117]. Following 2-3 h of brain ischemia, the free CoA level declines. This diminishment with simultaneous decrease of the acid-soluble CoA fraction achieves maximal reduction within 24 h under continued conditions of reperfusion (reoxygenation). These results confirm significance of the CoA system in pathogenic mechanisms of reoxygenation-reperfusion syndrome development.

CoA is one of the fundamental metabolism factors, and its biosynthesis and catabolism are subject to rigid control on the cell level. Therefore, as a rule, changes of particular CoA forms may happen only under extreme conditions and after prolonged period of time, sufficient to cause imbalance in metabolism regulation systems in the cell. Such imbalance starts to influence the CoA system during ischemia no earlier than one hour after occlusion of arteria carotis.

Data on the key role of the CoA biosynthesis system in maintaining redox potential of the glutathione system, neuronal membrane stability and defense of nitroperoxide acyl-CoA gives rationale to the use of CoA precursors in treatment of ischemia and ischemiareperfusion-induced damages in the brain tissue.

## **6. Role of selenium in mechanisms of antioxidant protection for brain**

Selenium is an essential microelement in different brain functions [119-124]. Neuroprotective potential of selenium is realized through the expression of selenoproteins: glutathione peroxidase, thioredoxine reductases, methionine sulfoxide reductases, selenoproteins P and R, which participate in regulation of the oxidation-reduction state of the neurones and glial cells under both physiologic conditions, and during oxidation stress [125, 126, 128]. Selenium regulates antioxidative processes in the CNS, protects brain tissue from neurodegenerative injuries during Alzheimer and Parkinson diseases, prione diseases, has antiischemic and angiogenic actions, etc. Insufficient level of selenium intensifies damages of neuron functions and structure caused by different endogenic and exogenic affections and leads to some neurodegenerative pathologies [122, 129-132].

The biological role of selenium is explained by the selenium presence in active sites of selenium-related enzymes [121, 133], which protect brain tissue during oxidative stress. Expression extremely diverse Se-containig proteins is observed in the brain. Selenoprotein P is required for transfer of selenium into the brain, and the brain selenium level is strictly dependent from an expression of selenoprotein [129]. Activity of Se-dependent enzymes in the brain is maintained at rather stable levels even during profound selenium deficiency, owing to the presence of unique Se-transport system in the brain (proteins containing selenium-cysteine, Se-transported protein of a Golgi apparatus). This system achieves its maximum value in hypothalamus.

Injections of selenium-containing compounds lead to an increase of activities of glutathione peroxidase and thioredoxin reductase, decrease of lipoperoxidation processes, cell defense from apoptosis [122, 126, 127]. Selenium ions activate oxidative-reductive enzymes of mitochondria and microsomes, take part in ATP synthesis, in electron transfer from hemoglobin to oxygen, maintain cysteine turnover, enhance α-tocoferol action.

## **7. Metabolic approaches to correction for brain ischemia-reperfusioncaused injuries**

Steady advances in the neurosciences have elicidated the pathophysiological mechanisms of brain ischemia and have suggested many therapeutic approaches to achieve neuroprotection in the acutely ischemic brain that are directed at specific injury mechanisms [134-136]. Nevertheless, methods of protection of ischemia and reperfusion-induced damages are still lacking [ 51, 137, 138]. Search for new ways of neurodefense during brain ischemia-reperfusion is necessary due to the absence of sufficient protective activity in the most substances with specific focus in clinical conditions: controlling excitotoxic effects of neurotransmitter amino acids (modulators of glutamic acid receptor activity and Cachannels), regulating redox status of cells, as well as presence of high toxicity in the most anti-ischemic medicines [51, 139-141]. In the past two decades, numerous attempts were made to use different substances with the effect on Ca level in a cell and glutamate extracellular level, aiming to apply these as drugs for ischemia-induced injuries treatment, but they have not been successful in men [51, 139-141]. For example, in experimental models, blockators of NMDA- and AMPA-receptors of glutamate exhibit high protective action, but they have strong side-effects and weak protective effects in humans, especially blockators of NMDA-receptors. The role of glutamate in neurotoxic phenomena during ischemia is known to be significant, but usage of glutamate receptor antagonists is rather problematic [51]. There are ongoing studies of Mg substances which block NMDAreceptors, as well as with blockators of AMPA-receptors. There have been recent proposals to combine usage of several drugs with different mechanisms of action. All of the above drugs have a common property – rather high toxicity. Therefore, usually a certain combination of drugs is applied in order to minimize their toxicity and maximize effectiveness [140].

502 Lipid Peroxidation

maximum value in hypothalamus.

**caused injuries** 

from neurodegenerative injuries during Alzheimer and Parkinson diseases, prione diseases, has antiischemic and angiogenic actions, etc. Insufficient level of selenium intensifies damages of neuron functions and structure caused by different endogenic and exogenic

The biological role of selenium is explained by the selenium presence in active sites of selenium-related enzymes [121, 133], which protect brain tissue during oxidative stress. Expression extremely diverse Se-containig proteins is observed in the brain. Selenoprotein P is required for transfer of selenium into the brain, and the brain selenium level is strictly dependent from an expression of selenoprotein [129]. Activity of Se-dependent enzymes in the brain is maintained at rather stable levels even during profound selenium deficiency, owing to the presence of unique Se-transport system in the brain (proteins containing selenium-cysteine, Se-transported protein of a Golgi apparatus). This system achieves its

Injections of selenium-containing compounds lead to an increase of activities of glutathione peroxidase and thioredoxin reductase, decrease of lipoperoxidation processes, cell defense from apoptosis [122, 126, 127]. Selenium ions activate oxidative-reductive enzymes of mitochondria and microsomes, take part in ATP synthesis, in electron transfer from

hemoglobin to oxygen, maintain cysteine turnover, enhance α-tocoferol action.

**7. Metabolic approaches to correction for brain ischemia-reperfusion-**

Steady advances in the neurosciences have elicidated the pathophysiological mechanisms of brain ischemia and have suggested many therapeutic approaches to achieve neuroprotection in the acutely ischemic brain that are directed at specific injury mechanisms [134-136]. Nevertheless, methods of protection of ischemia and reperfusion-induced damages are still lacking [ 51, 137, 138]. Search for new ways of neurodefense during brain ischemia-reperfusion is necessary due to the absence of sufficient protective activity in the most substances with specific focus in clinical conditions: controlling excitotoxic effects of neurotransmitter amino acids (modulators of glutamic acid receptor activity and Cachannels), regulating redox status of cells, as well as presence of high toxicity in the most anti-ischemic medicines [51, 139-141]. In the past two decades, numerous attempts were made to use different substances with the effect on Ca level in a cell and glutamate extracellular level, aiming to apply these as drugs for ischemia-induced injuries treatment, but they have not been successful in men [51, 139-141]. For example, in experimental models, blockators of NMDA- and AMPA-receptors of glutamate exhibit high protective action, but they have strong side-effects and weak protective effects in humans, especially blockators of NMDA-receptors. The role of glutamate in neurotoxic phenomena during ischemia is known to be significant, but usage of glutamate receptor antagonists is rather problematic [51]. There are ongoing studies of Mg substances which block NMDAreceptors, as well as with blockators of AMPA-receptors. There have been recent proposals to combine usage of several drugs with different mechanisms of action. All of the above

affections and leads to some neurodegenerative pathologies [122, 129-132].

Substances for metabolic therapy may be particularly useful during treatment of brain blood circulation injuries in the case of their simultaneous application with specific medicines because they have no toxic effects and may be used safely for prolonged period [51, 142, 143]. Apart from these drugs, compounds for so-called restoration therapy may be used. Their effects include restoration of metabolism and blood flow in damaged region. Application of the metabolic substances that help to maintain energy metabolism and redox status of glutathione system may be useful for remedying damages to the brain after ischemia-reperfusion [51, 143]. Previously we have shown high efficacy of pantothenic acid derivatives – CoA precursors, as a means of protecting cell membranes from different types of oxidative stress [117, 118, 144]. D-panthenol presents an important substance in this respect because it penetrates into the brain through blood-brain barrier easily and is converted into pantothenic acid, 4-phosphopantothenic acid, CoA, and after that into acyl-CoA (acetyl-, malonyl-, succinyl-CoA), which have high metabolic activity. These effects create the preconditions for stabilization of CoA-dependent processes of membrane phospholipid biosynthesis, neurotransmitter biosynthesis, regulation of energic processes, etc [118].

The efficacy of panthenol as a neuroprotector within a stroke model in rats has been demonstrated [117, 118, 144]. Panthenol not only decreased the volume of infarction, but also diminished neurological deficiency in animals [103]. Fairly high protector activity of Dpanthenol was observed in respect to changes of energic metabolism and glutathione system activity during brain ischemia-reperfusion. Protective effects of pantothenic derivatives is not related to their action as free radical scavengers, however. They act primarily as CoA precursors, whereas CoA accelerates various metabolic pathways, such as biosynthesis of glutathione, which constitutes one of the main systems of cell protection against oxidative stress.

Succinic acid is essential for keeping energy formation processes stable in the brain under extreme conditions [94-97]. Consequently, injections of panthenol and succinate following brain ishemia-reperfusion stabilize levels of lipoperoxidation in blood and in brain hemispheres, stabilize levels of protein SH-groups in blood, lead to significant decrease of the GSSG level and normalization of glutathione enzyme activities, as well as glutamate and glutamine metabolism in the brain to control values [118]. D-panthenol and succinate ammonium injection served to partially remedy the injuries and restore these parameters to their normal levels, especially if administered together. These effects are likely linked to activation of succinyl-CoA biosynthesis.

Attempts were made to use selenium-containing compounds for prevention of ischemiaindused injuries, such as ebselen (2-phenyl-1,2-benzisoselenozol-3), which imitated glutathione peroxidase activity [146-148]. However, under clinical conditions the ebselen was not effective. Di-(3-methylpyrazolil-4)-selenide (selecor) imitates effects of selenoproteins, has low toxicity, and satisfactory bioavailability. Additional injections of selecor increase effects of the panthenol and succinate, especially on the lipoperoxidation parameters and activities of glutathione system and selenium-bound enzymes, on ischemiareperfusion- induced injuries [149].

Effects of D-panthenol and succinate on decrease of lipoperoxidation activities contribute to the overall protective effects of the composition. However, it is evident that metabolic actions of the substances are related to their capasity for regulation of energy metabolism and mitochondrial respiration activity, restoration of the CoA-SH level and cell redox status, membrane-protective activity of the panthenol [118]. Addition of di-(3-methylpyrazolil-4) selenide (selecor) to D-panthenol and succinate does has limited effect on protective antioxidant properties of the composition. It is likely that this provides additional evidence for significance of specifically metabolic effects of the composition. Increase of selenium level in blood plasma, which may contribute in maintaining of antioxidant activity of glutathione system, takes place in the absence of selenoprotein substrates, after injections of panthenol and succinate. Nevertheless, addition of a selenium source to panthenol and succinate strengthened protective potential of the substances with respect to changes for enzyme activities of glutamate and glutamine which play an important role in maintaining of energy supply and detoxication in ischemic brain tissue and confirms the antiischemic effect of the substances. Effects of di-(3-methylpyrazolil-4)-selenide may be explained less by selenium supply as a selenoprotein component rather than by its modeling of selenoprotein activity, as is known to be the case with ebselen [146-148].

Therefore it is expected that the tested substances, such as panthenol, succinic acid, selecor, and potentially other metabolic therapy drugs may have high efficacy as neuroprotectors in brain ischemia and reperfusion-induced damages.

## **Author details**

Nina P. Kanunnikova and Natalya Z. Bashun *Department of Zoology and Physiology of Men and Animals, Yanka Kupala's Grodno State University, Grodno, Republic of Belarus* 

#### Andrey G. Moiseenok

*National Center for Foodstuffs, National Academy of Sciences of Belarus, Minsk, Republic of Belarus* 

### **8. References**


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504 Lipid Peroxidation

reperfusion- induced injuries [149].

activity, as is known to be the case with ebselen [146-148].

brain ischemia and reperfusion-induced damages.

*Department of Zoology and Physiology of Men and Animals,* 

*Yanka Kupala's Grodno State University, Grodno, Republic of Belarus* 

stroke. Acute ischemic stroke. Ed.JCG Rodriguez: 29-58.

Nina P. Kanunnikova and Natalya Z. Bashun

**Author details** 

Andrey G. Moiseenok

tissue. Stroke. 14: 329-331.

**8. References** 

selenoproteins, has low toxicity, and satisfactory bioavailability. Additional injections of selecor increase effects of the panthenol and succinate, especially on the lipoperoxidation parameters and activities of glutathione system and selenium-bound enzymes, on ischemia-

Effects of D-panthenol and succinate on decrease of lipoperoxidation activities contribute to the overall protective effects of the composition. However, it is evident that metabolic actions of the substances are related to their capasity for regulation of energy metabolism and mitochondrial respiration activity, restoration of the CoA-SH level and cell redox status, membrane-protective activity of the panthenol [118]. Addition of di-(3-methylpyrazolil-4) selenide (selecor) to D-panthenol and succinate does has limited effect on protective antioxidant properties of the composition. It is likely that this provides additional evidence for significance of specifically metabolic effects of the composition. Increase of selenium level in blood plasma, which may contribute in maintaining of antioxidant activity of glutathione system, takes place in the absence of selenoprotein substrates, after injections of panthenol and succinate. Nevertheless, addition of a selenium source to panthenol and succinate strengthened protective potential of the substances with respect to changes for enzyme activities of glutamate and glutamine which play an important role in maintaining of energy supply and detoxication in ischemic brain tissue and confirms the antiischemic effect of the substances. Effects of di-(3-methylpyrazolil-4)-selenide may be explained less by selenium supply as a selenoprotein component rather than by its modeling of selenoprotein

Therefore it is expected that the tested substances, such as panthenol, succinic acid, selecor, and potentially other metabolic therapy drugs may have high efficacy as neuroprotectors in

*National Center for Foodstuffs, National Academy of Sciences of Belarus, Minsk, Republic of Belarus* 

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## **Lipid Peroxidation and Polybrominated Diphenyl Ethers – A Toxicological Perspective**

Mary C. Vagula and Elisa M. Konieczko

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50345

## **1. Introduction**

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oxidative damage in focal cerebral ischemia. Free Rad.Biol.Med. 34: 56-63.

Understanding the structure and composition of plasma membrane is important as it guards the integrity and function of the cell as a whole. Lipid peroxidation is a process in which the lipids of the cell, particularly the membrane lipids, are degraded by oxidation resulting in the disruption of the entire cell. All cells are enclosed by a plasma membrane which serves as a boundary and it is the primary barrier between cell's internal and external environments. It is semi-permeable and plays a major role in regulating the transport of molecules into and out of the cells. This property of the plasma membrane is known as selective permeability. The fluid mosaic model describes the arrangement of the lipids and the proteins in the plasma membrane [1]. The lipids are smaller in size than the proteins. However, there are many more lipids than there are proteins. The lipids give the membranes their basic shape. Membrane lipids are amphipathic molecules, with hydrophilic and hydrophobic ends [2]. These lipids arrange in two layers (bilayer) such that the hydrophobic ends of one layer touches the hydrophobic ends of the other lipid layer. The hydrophilic ends of the phospholipids are in contact with the aqueous external and internal environments. Membrane lipids include phospholipids, cholesterol, sphingomyelin, triacylglycerides and glycolipids [3]. Several different types of phospholipids are found in cellular membranes: phosphatidylcholine (the most abundant), phosphatidylserine, phosphatidylinositol and phosphatidylethanolamime [4]. Each species of organisms has its own unique combination of these different phospholipids in its cellular membranes.

The proteins of the membrane float in the lipid bilayer. There are fewer proteins than lipids in the plasma membrane. However, they are much bigger than the lipids. The proteins can be associated with the lipids of the bilayer in one of the three ways [5]. Integral proteins are amphipathic molecules that span the entire width of lipid bilayer. Their hydrophilic and hydrophobic portions allow them to interact with both lipids and the aqueous environments

on either side of the lipids. Membrane associated proteins are associated with either external or internal surfaces of the membrane. These proteins often contain a lipid anchor which helps to hold them in the membrane. Peripheral proteins are attached to the inner surface of the membrane through covalent bonds to another protein. For example, the proteins that link the plasma membrane to the cytoskeleton. Plasma membrane proteins have several functions [6]. They serve as transporters, receptors, enzymes and as anchors, tying the cell to the external environment or to other cells. Carbohydrates attached to some proteins serve as identification markers and help in the differentiation of self from non-self cells [7].

The composition and structure of the plasma membrane described above is affected by free radicals. So the next section deals with free radicals, on how they are generated in the cells and their effects on cellular macromolecules.

## **2. Free radicals and Reactive Oxygen Species (ROS)**

A free radical may be defined as any atom or molecule that has one or more unpaired electrons in its outermost shell. Free radicals were discovered in biological material about 50 years ago [8] and since then, they have generated great curiosity for their involvement in disease and aging. Oxygen free radicals or reactive oxygen species (ROS) are defined as any free radical molecules containing oxygen. Some examples of the most common ROS include: hydrogen peroxide (H2O2), hydroxyl (•OH), and superoxide (O2• –) anion. Under normal conditions, ROS are produced as necessary intermediates in white blood cells to defend against invading pathogens, and they also play a role in intracellular signaling. Owing to their unpaired electrons in their outermost shell, ROS are highly reactive and unstable and they exert damage on biological structures. Free radicals react quickly with other molecules and "steal" electrons to acquire stability. The molecule which lost its electron to a free radical will become a free radical itself, thus beginning a chain reaction. Free radicals modify the structure of other molecules and cause oxidative damage. Once the process is started, if not quenched in time, it can eventually result in cell damage. Additionally, excessive amounts of free radicals are generated when tissues are damaged, for example, in times of tissue hypoxia, exposure to smoke, ultraviolet radiation and pollutants.

#### **2.1. Generation of reactive oxygen species**

Most of the energy required to fuel metabolic functions of aerobic organisms is produced at the cellular level in the mitochondria via the electron transport chain. In addition to energy, reactive oxygen species (ROS) are also generated from electron transport chain as part of normal cellular metabolic reactions. In this transport chain, ROS are formed at the mitochondrial inner membrane. Under normal conditions, the oxygen molecule acts as a terminal electron acceptor in the electron transport chain and is reduced to form water. However, sometimes electrons leak prematurely and reduce oxygen to form ROS. The electron transfer between ubiquinone-cytochrome b is the most probable site of ROS formation [9]. Microsomes and membranes of the nucleus also involve electron transportation through the cytochromes P450 and B5 which can produce free radicals [10]. The following few paragraphs discuss the generation of various ROS.

#### **2.2. Superoxide anion (O2• –)**

516 Lipid Peroxidation

on either side of the lipids. Membrane associated proteins are associated with either external or internal surfaces of the membrane. These proteins often contain a lipid anchor which helps to hold them in the membrane. Peripheral proteins are attached to the inner surface of the membrane through covalent bonds to another protein. For example, the proteins that link the plasma membrane to the cytoskeleton. Plasma membrane proteins have several functions [6]. They serve as transporters, receptors, enzymes and as anchors, tying the cell to the external environment or to other cells. Carbohydrates attached to some proteins serve as

The composition and structure of the plasma membrane described above is affected by free radicals. So the next section deals with free radicals, on how they are generated in the cells

A free radical may be defined as any atom or molecule that has one or more unpaired electrons in its outermost shell. Free radicals were discovered in biological material about 50 years ago [8] and since then, they have generated great curiosity for their involvement in disease and aging. Oxygen free radicals or reactive oxygen species (ROS) are defined as any free radical molecules containing oxygen. Some examples of the most common ROS include: hydrogen peroxide (H2O2), hydroxyl (•OH), and superoxide (O2• –) anion. Under normal conditions, ROS are produced as necessary intermediates in white blood cells to defend against invading pathogens, and they also play a role in intracellular signaling. Owing to their unpaired electrons in their outermost shell, ROS are highly reactive and unstable and they exert damage on biological structures. Free radicals react quickly with other molecules and "steal" electrons to acquire stability. The molecule which lost its electron to a free radical will become a free radical itself, thus beginning a chain reaction. Free radicals modify the structure of other molecules and cause oxidative damage. Once the process is started, if not quenched in time, it can eventually result in cell damage. Additionally, excessive amounts of free radicals are generated when tissues are damaged, for example, in times of

Most of the energy required to fuel metabolic functions of aerobic organisms is produced at the cellular level in the mitochondria via the electron transport chain. In addition to energy, reactive oxygen species (ROS) are also generated from electron transport chain as part of normal cellular metabolic reactions. In this transport chain, ROS are formed at the mitochondrial inner membrane. Under normal conditions, the oxygen molecule acts as a terminal electron acceptor in the electron transport chain and is reduced to form water. However, sometimes electrons leak prematurely and reduce oxygen to form ROS. The electron transfer between ubiquinone-cytochrome b is the most probable site of ROS formation [9]. Microsomes and membranes of the nucleus also involve electron transportation through the cytochromes P450 and B5 which can produce free radicals [10].

identification markers and help in the differentiation of self from non-self cells [7].

and their effects on cellular macromolecules.

**2. Free radicals and Reactive Oxygen Species (ROS)** 

tissue hypoxia, exposure to smoke, ultraviolet radiation and pollutants.

The following few paragraphs discuss the generation of various ROS.

**2.1. Generation of reactive oxygen species** 

When ground state oxygen accepts a single electron a superoxide anion (O2• - ) is formed.

$$\text{Oz +e} \rightarrow \text{Oz\*}-$$

Under normal physiological conditions cell organelles, namely, mitochondria, microsomes and peroxisomes, generate O2• - . The half-life of superoxide anion is longer than that of another potent ROS, hydroxyl radical (OH•), and it can react with biological molecules for longer times [10]. Phagocytic cells produce superoxide during a phenomenon known as the "respiratory burst" which occurs when they encounter pathogens. Phagocytic cells known to produce superoxide anions are macrophages, monocytes, neutrophils and eosinophils [11]. Superoxide anions can trigger the formation of hydroxyl and peroxyl radicals, which in an acidic environment can form hydrogen peroxide (H2O2).

#### **2.3. Hydrogen peroxide (H2O2)**

The addition of a second electron to O2• gives rise to the peroxide ion, O22-. At physiological pH any peroxide ion formed is protonated to hydrogen peroxide (H2O2).

$$2\text{O}\_2\cdot + 2\text{H}^+ \to \text{H}\_2\text{O}\_2 + \text{O}\_2$$

Hydrogen peroxide can then diffuse far from the site of its production to other sites where its biological effects may be greater. The diffusion range is extended by H2O2 carriers formed spontaneously by hydrogen bonding with compounds such as amino and dicarboxylic acids, peptides, proteins, nucleic acid bases, and nucleosides. Equilibrium exists between an adduct-forming compound and H2O2. The hydrogen peroxide adducts (HPAs) retard the decomposition of H2O2 up to several hundredfold [12]. The overall charge on an HPA influences the cytotoxic and clastogenic effects of H2O2. The adducts, especially L- Histidine, play an important role in the stabilization and reduction in the reactivity of the hydrogen peroxide thereby preventing single strand breaks of the DNA in cell free DNA systems [13].

#### **2.4. Hydroxyl radical (•OH)**

The hydroxyl radical (•OH) can be formed by the homolytic fission of the O-O bond of the H2O2 molecule. Simple mixing of iron (II) salts with the H2O2 also forms the •OH radical. This reaction was first reported by Fenton in 1894 and it is called Fenton's reaction. The hydroxyl radicals can also be produced by Haber-weiss reactions in the cells, in which H2O2 interacts with superoxide radical [10]. Additionally, copper salts can also react with H2O2 to generate •OH radicals. All the three ways of •OH radical generation is shown below:

By the decomposition of H2O2 by Fenton's reaction:

$$\mathrm{Fe^{2+} + HxO\_2 \to Fe^{3+} + OH + OH^-}$$

and H2O2 interaction with superoxide radical by the Haber-Weiss reaction:

$$\text{O2} \cdot \text{+ HxO} \cdot \text{H} \cdot \text{O2} \cdot \text{+ HxO} \cdot \text{+OH}$$

The hydroxyl radical can also be formed when copper salts react with H2O2 to make hydroxyl radicals as shown below:

$$\mathrm{Cu^{+} + H\_{2}O\_{2} \rightarrow Cu^{2+} + OH^{+} + OH^{-}}$$

Although the hydroxyl radical (OH<sup>∙</sup> ) has the shortest half-life, it is one of the most reactive of all ROS [14, 15]. Due to this property it reacts with almost all macromolecules found in the cells such as proteins, nucleic acids (RNA and DNA), phospholipids, sugars and carbohydrates, and exerts deleterious effects on them.

#### **2.5. Free radicals and the damage to cellular components**

Excess generation of free radicals combined with the failure of cells to scavenge them through protective mechanisms (antioxidative agents and enzymes) results in oxidative damage. Oxidative damage is witnessed in the form of free radical mediated attack on macromolecules within their vicinity and causing disruption of these molecules. Severe oxidative stress can even result in apoptosis and cell death [16]. Free radical mediated damage to proteins, carbohydrates, nucleic acids and lipids is described below.

#### **2.6. Free radical damage to proteins**

The oxidization reaction of free radicals on amino acids causes changes in the physical properties of proteins that they compose. These physical changes are of three types: fragmentation, aggregation and susceptibility to enzyme digestion [17]. The fragmentation of albumin and collagen due to free radical mediated oxidization is a classic example of this phenomenon [18, 19]. Selective fragmentation at specific amino acids, especially at proline, histidine and arginine, is observed due to their close association with transition metals. The second type of physical change, protein aggregation, is believed to be caused predominantly by hydroxyl radicals due to their ability to form cross-links between the constitutive amino acids. Lastly, gross alteration in the conformation of protein structure may be the reason for the susceptibility of these altered proteins to enzyme digestion [10].

#### **2.7. Free radical damage to carbohydrates**

Hydroxyl radicals generated by Fenton reaction are reported to induce damage on simple carbohydrates [20]. It is also reported that oxidation of glucose may be a means of quenching hydroperoxide radicals while also generating a source of oxygen free radicals [21]. Another study indicated that rapid auto-oxidation of glucose results in the formation of dicarbonyl and H2O2 under physiological conditions [22]. Thus, oxidized glucose can react with proteins in a process called glucosylation or glucation leading to the disruption in the function of these macromolecules. Glucosylation of certain long-lived proteins by a nonenzymic reaction with free glucose may contribute to ageing.

#### **2.8. Free radicals and the alteration of gene expression**

518 Lipid Peroxidation

hydroxyl radicals as shown below:

Although the hydroxyl radical (OH<sup>∙</sup>

**2.6. Free radical damage to proteins** 

carbohydrates, and exerts deleterious effects on them.

**2.5. Free radicals and the damage to cellular components** 

the susceptibility of these altered proteins to enzyme digestion [10].

enzymic reaction with free glucose may contribute to ageing.

**2.7. Free radical damage to carbohydrates** 

O2∙-

+ H2O2 →O2 +H2O+**<sup>∙</sup>**

The hydroxyl radical can also be formed when copper salts react with H2O2 to make

of all ROS [14, 15]. Due to this property it reacts with almost all macromolecules found in the cells such as proteins, nucleic acids (RNA and DNA), phospholipids, sugars and

Excess generation of free radicals combined with the failure of cells to scavenge them through protective mechanisms (antioxidative agents and enzymes) results in oxidative damage. Oxidative damage is witnessed in the form of free radical mediated attack on macromolecules within their vicinity and causing disruption of these molecules. Severe oxidative stress can even result in apoptosis and cell death [16]. Free radical mediated

The oxidization reaction of free radicals on amino acids causes changes in the physical properties of proteins that they compose. These physical changes are of three types: fragmentation, aggregation and susceptibility to enzyme digestion [17]. The fragmentation of albumin and collagen due to free radical mediated oxidization is a classic example of this phenomenon [18, 19]. Selective fragmentation at specific amino acids, especially at proline, histidine and arginine, is observed due to their close association with transition metals. The second type of physical change, protein aggregation, is believed to be caused predominantly by hydroxyl radicals due to their ability to form cross-links between the constitutive amino acids. Lastly, gross alteration in the conformation of protein structure may be the reason for

Hydroxyl radicals generated by Fenton reaction are reported to induce damage on simple carbohydrates [20]. It is also reported that oxidation of glucose may be a means of quenching hydroperoxide radicals while also generating a source of oxygen free radicals [21]. Another study indicated that rapid auto-oxidation of glucose results in the formation of dicarbonyl and H2O2 under physiological conditions [22]. Thus, oxidized glucose can react with proteins in a process called glucosylation or glucation leading to the disruption in the function of these macromolecules. Glucosylation of certain long-lived proteins by a non-

damage to proteins, carbohydrates, nucleic acids and lipids is described below.

Cu+ + H2O2→Cu2++ **<sup>∙</sup>**

OH

OH+ OH-

) has the shortest half-life, it is one of the most reactive

Free radicals are extensively implicated for their role in DNA damage and alteration in the gene expression leading to various types of cancers [23]. It has been reported that about twenty types of changes are induced by ROS in the DNA. The damage in the DNA's deoxyguanosine residues is estimated to be in the range of 8 to 83 residues per million deoxyguanosine residues. This damage increases with age and seems to affect organs such as the liver, kidney and spleen while leaving brain tissue relatively undamaged [24]. Mitochondrial DNA experiences greater damage due to proximity to the ROS generated from the electron transport chain of mitochondrial membranes. Free radical induced damage to DNA includes a range of specifically oxidized purines and pyrimidines, alkali labile sites, single strand breaks and instability formed directly or by repair processes [25,26]. The pyrimidine residues, cytosine and thymine, of DNA are the most susceptible to the attack by hydroxyl radical, followed in decreasing levels of susceptibility by adenine, guanine and deoxyribose sugar [10]. Some of these modified bases have been found to possess mutagenic properties. Therefore, if not repaired they can lead to carcinogenesis.

#### **2.9. Free radicals and lipid peroxidation**

Free radicals steal electrons from the cell membrane's polyunsaturated fatty acids (PUFAs) and initiate an attack called lipid peroxidation. The peroxidation of lipids occurs in three steps, namely, initiation, propagation and termination. Free radicals target the carboncarbon double bond of PUFAs, as this double-bond weakens the carbon-hydrogen bond, permitting easy dissociation of the hydrogen by a free radical. During the first step, a single electron is stripped by a free radical from hydrogen atoms associated with the carbon at the double-bond leaving the carbon with a single electron. Molecular arrangements occur in an attempt to stabilize, leading to the formation of conjugated dienes. In the propagation phase, the conjugated diene readily reacts with molecular oxygen and transitions to a hydroperoxide radical that begins stealing electrons from other lipid molecules, resulting in a lipid hydroperoxide [11]. The hydroperoxide molecules undergo decomposition with the aid of transition metals into alcoxyl and peroxyl radicals that, in turn, can initiate a chain reaction and further propagate lipid peroxidation and cause damage to the cell. The three phases or steps of lipid peroxidation are shown in the following equations:

> 2 2 2 2 2 Initiation reaction Lipid H OH Lipid H O Hydroperoxide formation Lipid O Lipid-O Lipid hydroperoxide Lipid-O Lipid H Lipid Lipid-O H

The important fatty acids that undergo peroxidation are: linoleic acid, arachidonic acid, and docosahexaenoic acids [14]. The hydroxyl radical damages cell membranes and other lipoproteins by lipid peroxidation. It is important to note that the low density lipoprotein based lipid peroxidation plays a significant role in atherosclerosis [27].

The generation of free radicals and their action on macromolecules thus leads to disruption of cellular functions. The following section discusses chemicals that are present in the body to protect against these damaging radicals.

## **3. Antioxidative protection against reactive oxygen species (Free radicals)**

All aerobic cells have an armory of chemicals known as antioxidants which are capable of counteracting the damaging effects of oxidative free radicals and inhibiting the oxidation of other molecules in the body. Based on the mechanism of their action, these antioxidants are divided into two categories: chain breaking antioxidants and preventive antioxidants. Vitamin E, glutathione, vitamin C and beta-carotene are some examples of the chain breaking type as they donate their own electron to the existing free radical and prevent the continuation of oxidation chain reaction. These antioxidants are found in the plasma and interstitial fluid [28, 29]. The preventative category of antioxidants consists of enzymes that scavenge the free radicals before they initiate an oxidative attack.

Another way to categorize these antioxidants is based on their solubility in lipids or water. The antioxidants which are lipid soluble are located in the cellular membrane and lipoproteins, while the water soluble ones are located in the aqueous environments, such as the cytoplasm inside cells and the blood [30]. The following few sections discuss some of the important antioxidants of cells.

#### **3.1. Glutathione**

Glutathione (GSH) is one of the important cellular antioxidants. It is made up of three amino acids namely, cysteine, glutamic acid and glycine. All the cells of the body have the ability to synthesize glutathione, but it is found in high concentrations in the liver, lungs, and intestinal tract. The important functions of the glutathione include: anti-oxidation, detoxification, strengthening the immune system and signal transduction under stressful conditions. The diverse functions of GSH are due to the sulfhydryl group in cysteine, enabling it to chelate and detoxify harmful substances [31]. Additionally, glutathione also plays a role in processing medications and cancer-causing compounds (carcinogens), and in building DNA, proteins, and other important cellular components. Its usefulness as an antioxidative agent lies in its ability to donate electrons, and in that process it is oxidized to glutathione disulfide (GSSG) form. By donating an electron to a free radical, GSH can quench the unstable and highly reactive free radical and thus can prevent free radical mediated oxidative damage in the cells. GSSG is reduced to GSH by NADPH, an electron donor in this reaction. This reduction reaction is catalyzed by glutathione reductase. Thus the ratio between reduced and oxidized glutathione serves as an indicator of cellular toxicity mediated by oxidative damage. Large amounts of GSH are produced and stored in the liver, where it is used to detoxify harmful compounds. GSH levels are found to decline with age leaving the body susceptible to the damage by free radicals.

#### **3.2. Vitamin C, vitamin E and beta carotene**

520 Lipid Peroxidation

lipoproteins by lipid peroxidation. It is important to note that the low density lipoprotein

The generation of free radicals and their action on macromolecules thus leads to disruption of cellular functions. The following section discusses chemicals that are present in the body

**3. Antioxidative protection against reactive oxygen species (Free radicals)** 

All aerobic cells have an armory of chemicals known as antioxidants which are capable of counteracting the damaging effects of oxidative free radicals and inhibiting the oxidation of other molecules in the body. Based on the mechanism of their action, these antioxidants are divided into two categories: chain breaking antioxidants and preventive antioxidants. Vitamin E, glutathione, vitamin C and beta-carotene are some examples of the chain breaking type as they donate their own electron to the existing free radical and prevent the continuation of oxidation chain reaction. These antioxidants are found in the plasma and interstitial fluid [28, 29]. The preventative category of antioxidants consists of enzymes that

Another way to categorize these antioxidants is based on their solubility in lipids or water. The antioxidants which are lipid soluble are located in the cellular membrane and lipoproteins, while the water soluble ones are located in the aqueous environments, such as the cytoplasm inside cells and the blood [30]. The following few sections discuss some of the

Glutathione (GSH) is one of the important cellular antioxidants. It is made up of three amino acids namely, cysteine, glutamic acid and glycine. All the cells of the body have the ability to synthesize glutathione, but it is found in high concentrations in the liver, lungs, and intestinal tract. The important functions of the glutathione include: anti-oxidation, detoxification, strengthening the immune system and signal transduction under stressful conditions. The diverse functions of GSH are due to the sulfhydryl group in cysteine, enabling it to chelate and detoxify harmful substances [31]. Additionally, glutathione also plays a role in processing medications and cancer-causing compounds (carcinogens), and in building DNA, proteins, and other important cellular components. Its usefulness as an antioxidative agent lies in its ability to donate electrons, and in that process it is oxidized to glutathione disulfide (GSSG) form. By donating an electron to a free radical, GSH can quench the unstable and highly reactive free radical and thus can prevent free radical mediated oxidative damage in the cells. GSSG is reduced to GSH by NADPH, an electron donor in this reaction. This reduction reaction is catalyzed by glutathione reductase. Thus the ratio between reduced and oxidized glutathione serves as an indicator of cellular toxicity mediated by oxidative damage. Large amounts of GSH are produced and stored in the liver, where it is used to detoxify harmful compounds. GSH levels are found to decline with age

based lipid peroxidation plays a significant role in atherosclerosis [27].

scavenge the free radicals before they initiate an oxidative attack.

leaving the body susceptible to the damage by free radicals.

to protect against these damaging radicals.

important antioxidants of cells.

**3.1. Glutathione** 

Vitamin C is a water soluble antioxidant which plays an important role in protecting the body against oxidative damage. Chemically, it is a carbohydrate-like substance. Human beings, unlike other mammals, do not have the ability to synthesize Vitamin C. Therefore, it must be furnished through the diet. Vitamin C is easily oxidized and many functions in the body depend on this property. It stops the free radical chain reaction by donating an electron and prevents the reaction from progressing.

Carotenoids are a class of cellular antioxidants. Carotenoids are referred as provitamin A as they can potentially yield Vitamin A. Free radicals which can damage DNA and cause diseases like macular degeneration, coronary artery disease and cancer are neutralized by these carotenoids. Beta-carotene is perhaps the best known carotenoid. It was isolated in 1831 and its structure was determined in 1931.

Vitamin E is a fat soluble compound which also can impede oxidation to some degree [32]. Though there are eight forms in nature, only alpha-tocopherol form is functional in human beings. Vitamin E stops the production of ROS formation when fat undergoes oxidation. In addition to the above mentioned vitamins, essential trace elements, selenium, zinc, manganese, and copper, also play a critical role as antioxidants.

#### **3.3. Antioxidant enzymes**

Antioxidant enzymes inside the cells are the main defense against the free radicals. The most important antioxidant enzymes present inside the cells are superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and glutathione S-transferase (GST). SOD catalyzes the dismutation of superoxide anion to O2 and H2O2. This reaction is considered to be the body's primary defense against free radicals as it prevents further generation of free radicals [29]. SOD is found in high levels in the liver, adrenal glands, kidney and spleen [33]. GPx, catalyzes the reduction of hydroperoxides and serves to protect the cell from hostile free radicals. Catalase, another antioxidant enzyme, along with GPx, is essential for removing hydrogen peroxide formed during oxidation reactions, and is found in high levels in liver, kidney and red blood cells. Both catalase and GPx enzymes exhibit a great degree of cooperativity in their action. Glutathione S-Transferase (GST) is yet another antioxidant enzyme primarily responsible for cellular defense mechanism against ROS. GST metabolizes xenobiotics, whether they are of endogenous or exogenous origin [34]. GST along with glutathione and GPx neutralizes free radicals and lipid hydroperoxides especially at low levels of oxidative stress [35]. The undesirable end products of lipid peroxidation are thus detoxified by GST. All of these enzymes prove to be indispensable in the cellular antioxidant defense mechanism and they are all necessary for the survival of the cell, even in normal conditions. An appropriate equilibrium between antioxidant enzyme activities is vital to ensure the cell survival during increased oxidative stress.

However some exogenous factors, such as environmental contaminants, have the potential to disrupt this equilibrium and cause incredible damage to the cells. The following section describes a specific group of brominated flame retardants called polybrominated diphenyl ethers (PBDEs).

## **4. Polybrominated diphenyl ethers: A class of brominated flame retardants**

Fire accidents in the United States have the potential to cause severe economic problems for the individuals and for society at large. Each year, several thousand Americans die, several thousand more are injured, and millions of dollars of loss are all due to fires. To recover from these losses, individuals, their insurance companies, and society as a whole spend significant resources on temporary housing, groceries and meals, medical costs, psychological support, and relocation costs, to name a few expenditures. These ancillary costs may be 10-times higher than the cost to rebuild structures actually gutted by fire. To give a rough idea, the annual losses from natural calamities such as floods, hurricanes, tornadoes, earthquakes, etc., all put together in the United States equal just a fraction of total loss due to fires [36]. To comply with fire safety regulations in the United States manufacturers must follow strict guidelines in order to reduce the flammability of their products. For years, manufacturers have added chemicals to plastics and fabrics in order to reduce combustibility and increase overall safety of the products. Different types of brominated flame retardants have the promise of slowing down fire and hence are being extensively used. Figure 1 shows the chemical structures of four typical bromine compounds, namely, polybrominated biphenyls (PBBs), polybrominated diphenyl ethers (PBDEs), hexabromocyclododecane (HBCDs) and tetrabromobisphenol A (TBBPAs) [37].

**Figure 1.** Chemical structures of BFRs. (A) PBBs, (B) PBDEs, (C) HBCD, and (D) TBBPA [37]

In the past few decades, use of PBDEs has become quite commonplace and these chemicals are added in large quantities to many commercial and household products, such as computers, TVs, foam mattresses, and carpets, to inhibit ignition and prevent fire accidents [37]. At high temperatures PBDEs liberate bromine atoms, which are effective at hindering the basic oxidizing reactions that drive combustion. Ever since PBDEs were introduced into the market, deaths due to fire accidents dropped significantly. Functionally, these flame retardants are similar to polychlorinated biphenyls (PCBs) that were banned from production in the U.S in the late 1970s due to potential adverse health effects and a persistent bio-accumulative nature [38]. Like PCBs, the PBDEs class contains 209 possible congeners. During the synthesis of PBDEs, a bromination reaction occurs on the biphenyl rings as one or more bromine atoms are attached to the biphenyl rings giving rise to one of the possible 209 possible congeners of PBDEs. The maximum number of bromine atoms that can attach to biphenyl rings is 10 and this brominated flame retardant is called decaBDE. Commercially these chemicals are available in three mixtures, pentaBDEs, octaBDEs and decaBDEs. Congeners with 1-5 bromine atoms are referred as lower BDEs and those with 6 - 10 are referred as higher BDEs.

#### **4.1. Production of PBDEs**

522 Lipid Peroxidation

ethers (PBDEs).

**retardants** 

describes a specific group of brominated flame retardants called polybrominated diphenyl

Fire accidents in the United States have the potential to cause severe economic problems for the individuals and for society at large. Each year, several thousand Americans die, several thousand more are injured, and millions of dollars of loss are all due to fires. To recover from these losses, individuals, their insurance companies, and society as a whole spend significant resources on temporary housing, groceries and meals, medical costs, psychological support, and relocation costs, to name a few expenditures. These ancillary costs may be 10-times higher than the cost to rebuild structures actually gutted by fire. To give a rough idea, the annual losses from natural calamities such as floods, hurricanes, tornadoes, earthquakes, etc., all put together in the United States equal just a fraction of total loss due to fires [36]. To comply with fire safety regulations in the United States manufacturers must follow strict guidelines in order to reduce the flammability of their products. For years, manufacturers have added chemicals to plastics and fabrics in order to reduce combustibility and increase overall safety of the products. Different types of brominated flame retardants have the promise of slowing down fire and hence are being extensively used. Figure 1 shows the chemical structures of four typical bromine compounds, namely, polybrominated biphenyls (PBBs), polybrominated diphenyl ethers (PBDEs), hexabromocyclododecane (HBCDs) and tetrabromobisphenol A (TBBPAs) [37].

**4. Polybrominated diphenyl ethers: A class of brominated flame** 

**Figure 1.** Chemical structures of BFRs. (A) PBBs, (B) PBDEs, (C) HBCD, and (D) TBBPA [37]

In the past few decades, use of PBDEs has become quite commonplace and these chemicals are added in large quantities to many commercial and household products, such as computers, TVs, foam mattresses, and carpets, to inhibit ignition and prevent fire accidents [37]. At high temperatures PBDEs liberate bromine atoms, which are effective at hindering PBDEs were introduced into the market in the U.S. in the 1970s and peaked in the late 1990s. The production of various PBDEs in different regions of the world is shown in Table 1[37].


Data from BSEF (2001)

**Table 1.** BFRs produced (metric tons) in various regions of the world [37]

Table 2 shows the percent composition of various commercial PBDE mixtures. Currently, deca BDE constitutes 97% of the total production of PBDEs worldwide and it is the only BDE which is in use in the US [39].


**Table 2.** Percent composition of commercial PBDE flame retardant mixtures [39].

Many research findings indicate that lower brominated congeners manifest greater affinity for lipids and accumulation. Hence they are more toxic and pose a greater health risk to humans and livestock than the higher brominated congeners [40].

#### **4.2. Exposure to PBDEs**

PBDEs are lipophilic and resistant to chemical and physical degradation. Due to their persistent nature, they have become widespread environmental contaminants and about 97% of the American adult population has detectable levels of PBDEs [41]. Over the last 30 years the levels of these compounds in humans have increased and their levels in the US population are 10-100 times higher than levels measured in the Europe and Asia. According to a recent report, their levels in humans may be leveling off or decreasing due to their ban in many states of the US and European countries[39]. They are reported to be present in sediments, soil, outdoor and indoor air, household dust, foods, birds, fish, and terrestrial organisms [42].They have been detected in human serum, adipose tissue, and breast milk, and long-term exposure to these contaminants may pose a human health risk, especially to the developing fetus.

Despite their ban in many countries, vast amounts of these compounds are found to persist in existing consumer products, potentially contributing to environmental and human burdens for years to come. When released into the environment PBDEs adsorb rapidly onto solid particles such as soil or sediments due to their low water solubility and vapor pressure. Therefore, sediments and sewage sludge serve as a sink for these pollutants. These contaminants are still bioavailable even after adsorbing to the sediment particles [43]. PBDEs exposure to human being occurs through inhalation of household and workplace dust and by eating PBDE-contaminated foods. Exposure to PBDEs is nearly impossible to avoid as they are found in air, water and food. The fetus is also exposed to these compounds in utero, and after birth the newborn continues to be exposed from breast milk, where the toxins are transferred from the mother to the baby. A 25-year Swedish study found that the concentration of PBDEs in breast milk doubled every five years during the 25-year period [44]. The PBDE levels in breast milk from North American women are much higher than in breast milk from Swedish women, indicating greater exposure to PBDEs in this continent.

#### **4.3. Dietary intake of PBDEs**

The dietary intake of PBDEs from food in humans between ages 0-60 years are presented in Table 3. The intake of PBDEs was the highest in the first year during nursing. Breast milk has the highest levels of PBDEs. The intake of PBDEs per kg body weight reduced gradually with age. Children have higher PBDE intake than adults due to higher food intake per kg body weight [45].

#### **4.4. Adverse effects of PBDEs in laboratory animals**

Few toxic studies on PBDEs are reported in laboratory animals [46,47]. Some studies report these compounds to be toxic, carcinogenic and endocrine disruptors [48]. There are some reports indicating that PBDEs can induce disruption in spontaneous behavior, reduced habituation, impaired memory and learning in rats exposed during critical developmental period [46, 47]. BDE-99 is a penta congener and it has been found to induce hyperactivity and impaired spermatogenesis in rats. Additionally, two recent in-vitro studies showed the induction of oxidative stress in rat neurons exposed to BDE-47 (another congener of penta-BDE congener) in primary cultured rat hippocampal neurons [49] and human cells [50] serving as note-worthy examples that call for more careful assessment of these toxic substances.


**Table 3.** Daily intake of PBDEs from food (pg/kg body weight) [45]

#### **5. Lipid peroxidation during PBDE exposure**

524 Lipid Peroxidation

**4.2. Exposure to PBDEs** 

the developing fetus.

**4.3. Dietary intake of PBDEs** 

**4.4. Adverse effects of PBDEs in laboratory animals** 

body weight [45].

PBDEs are lipophilic and resistant to chemical and physical degradation. Due to their persistent nature, they have become widespread environmental contaminants and about 97% of the American adult population has detectable levels of PBDEs [41]. Over the last 30 years the levels of these compounds in humans have increased and their levels in the US population are 10-100 times higher than levels measured in the Europe and Asia. According to a recent report, their levels in humans may be leveling off or decreasing due to their ban in many states of the US and European countries[39]. They are reported to be present in sediments, soil, outdoor and indoor air, household dust, foods, birds, fish, and terrestrial organisms [42].They have been detected in human serum, adipose tissue, and breast milk, and long-term exposure to these contaminants may pose a human health risk, especially to

Despite their ban in many countries, vast amounts of these compounds are found to persist in existing consumer products, potentially contributing to environmental and human burdens for years to come. When released into the environment PBDEs adsorb rapidly onto solid particles such as soil or sediments due to their low water solubility and vapor pressure. Therefore, sediments and sewage sludge serve as a sink for these pollutants. These contaminants are still bioavailable even after adsorbing to the sediment particles [43]. PBDEs exposure to human being occurs through inhalation of household and workplace dust and by eating PBDE-contaminated foods. Exposure to PBDEs is nearly impossible to avoid as they are found in air, water and food. The fetus is also exposed to these compounds in utero, and after birth the newborn continues to be exposed from breast milk, where the toxins are transferred from the mother to the baby. A 25-year Swedish study found that the concentration of PBDEs in breast milk doubled every five years during the 25-year period [44]. The PBDE levels in breast milk from North American women are much higher than in breast milk from Swedish women, indicating greater exposure to PBDEs in this continent.

The dietary intake of PBDEs from food in humans between ages 0-60 years are presented in Table 3. The intake of PBDEs was the highest in the first year during nursing. Breast milk has the highest levels of PBDEs. The intake of PBDEs per kg body weight reduced gradually with age. Children have higher PBDE intake than adults due to higher food intake per kg

Few toxic studies on PBDEs are reported in laboratory animals [46,47]. Some studies report these compounds to be toxic, carcinogenic and endocrine disruptors [48]. There are some reports indicating that PBDEs can induce disruption in spontaneous behavior, reduced habituation, impaired memory and learning in rats exposed during critical developmental period [46, 47]. BDE-99 is a penta congener and it has been found to induce hyperactivity and Despite many studies, exact mechanism of toxicity of PBDEs is not yet clear and there is no definite explanation as to how PBDEs trigger the above mentioned deleterious effects. There appeared a school of thought that PBDEs, by reason of their electron affinity favor free radical formation in various tissues [51]. In the face of increased free radical generation the antioxidant enzymes namely, SOD, GPx, Catalase, and GST, are expected to give protection to the cells against the oxidative damage. An appropriate equilibrium between the antioxidant enzyme activities and free radicals is vital to ensure the cell's survival during increased oxidative stress. In the process of investigating the toxic mechanism authors have surmised oxidative stress induction in mice exposed to BDE-209, one of the most abundantly used flame retardant worldwide. This is based on a recent study showing the induction of oxidative stress in rat neurons exposed to BDE-47, a penta congener [49]. To interpret the adverse effects, both an assessment of the magnitude of free radical production and a careful evaluation of the threshold of protection in terms of antioxidant enzyme activity is necessary. Antioxidant enzymes such as SOD, GPx, GST, and catalase could also be the key indicators that could disclose the sequence of biochemical dysfunctions and alterations in signal transduction brought about by BDE-209. In the following few paragraphs the research protocols that one of the authors (MCV) adopted to investigate the biological markers for oxidative stress following BDE-209 exposure are explicated. The study is performed on the liver and brain tissues of adult mice. The liver is a major biotransforming organ and brain is the most sensitive target to lipid soluble toxicants. Studying these two organs would give a clear picture of how this particular BDE is handled by these two vital organs. Further, since the metabolism and excretion of many PBDEs or for that matter any xenobiotics are tissue, species, sex and age dependent [52] it is intended to understand the effects of BDE-209 on these two vital organs; thus the hepato- and neurotoxic mechanisms could be simultaneously studied and correlated.

## **5.1. Materials and methods**

#### *5.1.1. Chemicals*

BDE- 85- CS (certified standard) was procured from Cambridge Isotope laboratories, Inc., Andover, MA, USA. Assay kits for the determination of lipid hydroperoxides, SOD, GPx, catalase, and GST were purchased from Cayman Chemical Company, Ann Arbor, Michigan, USA. BCATM protein assay kit was purchased from Pierce Biotechnology Inc., Rockford IL, USA, for the protein assay of the sample. All other chemicals needed for the experiments were obtained from Sigma Aldrich Company.

#### *5.1.2. Animals*

Adult male Swiss Webster mice were obtained from Taconic Inc (Hudson, New York) and used for oxidative stress studies. Mice weighing 33±3 g were maintained at 230C±20C with 12-hour light/12-hour dark photoperiods. Mice were provided food pellets and water ad libitum and were allowed to acclimatize for 15 days. The guidelines set forth by the Institutional Animal Care and Use Committee, Gannon University, Erie, Pennsylvania, were followed in the maintenance and use of mice. Mice were grouped into two groups randomly (*n* = 9). Group 1 was injected intraperitoneally with 0.25mg/kg body weight of BDE-209 for four consecutive days and group 2 served as control and received same volume of vehicle solution, corn oil. Oral route of administration was not considered in this study as the aim of this work is to study the biochemical alterations induced at this particular concentration and administering via oral route results in elimination of some fraction of this chemical through feces. The present dosage of BDE-209 was selected based on some reports on other penta-BDE congeners in rodents [42,52]. In addition, the dosage used in the present study also is commensurate with the average levels of PBDEs (except BDE-209) found in household dust [53]. On the fifth day mice were sacrificed and liver and brain tissues were separated, washed in phosphate-buffered saline (PBS), and weighed. For all the biochemical assays, a 5% tissue homogenate of liver or brain was prepared using appropriate homogenizing buffers. The protocols that came with the assay kits were used for all biochemical assays and they are described below. The protein content of all the enzyme samples is assayed using Pierce® BCA Protein Assay Kit. Bovine serum albumin was used as standard.

#### *5.1.3. Lipid hydroperoxides*

Lipid peroxidation is quantified by a new method which directly quantifies the lipid hydroperoxides utilizing the redox reactions with ferrous ions.The liver and brain tissues from control and experimental mice were homogenized in nanopure water and the lipid hydroperoxides from the homogenate were extracted into chloroform. According to the protocol accompanying the lipid hydroperoxide assay kit, supplied by the Cayman Chemical Company, this chloroform extract was then used for the determination of lipid hydroperoxides.

#### *5.1.4. Superoxide Dismutase (SOD)*

526 Lipid Peroxidation

*5.1.1. Chemicals* 

*5.1.2. Animals* 

**5.1. Materials and methods** 

albumin was used as standard.

*5.1.3. Lipid hydroperoxides* 

hydroperoxides.

were obtained from Sigma Aldrich Company.

BDE- 85- CS (certified standard) was procured from Cambridge Isotope laboratories, Inc., Andover, MA, USA. Assay kits for the determination of lipid hydroperoxides, SOD, GPx, catalase, and GST were purchased from Cayman Chemical Company, Ann Arbor, Michigan, USA. BCATM protein assay kit was purchased from Pierce Biotechnology Inc., Rockford IL, USA, for the protein assay of the sample. All other chemicals needed for the experiments

Adult male Swiss Webster mice were obtained from Taconic Inc (Hudson, New York) and used for oxidative stress studies. Mice weighing 33±3 g were maintained at 230C±20C with 12-hour light/12-hour dark photoperiods. Mice were provided food pellets and water ad libitum and were allowed to acclimatize for 15 days. The guidelines set forth by the Institutional Animal Care and Use Committee, Gannon University, Erie, Pennsylvania, were followed in the maintenance and use of mice. Mice were grouped into two groups randomly (*n* = 9). Group 1 was injected intraperitoneally with 0.25mg/kg body weight of BDE-209 for four consecutive days and group 2 served as control and received same volume of vehicle solution, corn oil. Oral route of administration was not considered in this study as the aim of this work is to study the biochemical alterations induced at this particular concentration and administering via oral route results in elimination of some fraction of this chemical through feces. The present dosage of BDE-209 was selected based on some reports on other penta-BDE congeners in rodents [42,52]. In addition, the dosage used in the present study also is commensurate with the average levels of PBDEs (except BDE-209) found in household dust [53]. On the fifth day mice were sacrificed and liver and brain tissues were separated, washed in phosphate-buffered saline (PBS), and weighed. For all the biochemical assays, a 5% tissue homogenate of liver or brain was prepared using appropriate homogenizing buffers. The protocols that came with the assay kits were used for all biochemical assays and they are described below. The protein content of all the enzyme samples is assayed using Pierce® BCA Protein Assay Kit. Bovine serum

Lipid peroxidation is quantified by a new method which directly quantifies the lipid hydroperoxides utilizing the redox reactions with ferrous ions.The liver and brain tissues from control and experimental mice were homogenized in nanopure water and the lipid hydroperoxides from the homogenate were extracted into chloroform. According to the protocol accompanying the lipid hydroperoxide assay kit, supplied by the Cayman Chemical Company, this chloroform extract was then used for the determination of lipid The liver and brain tissues from control and experimental mice were homogenized in 4-(2 hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer containing 1 mmole/L ethylene glycol tetraacetic acid (EGTA), 210 mmole/L mannitol and 70 mmole/L sucrose and centrifuged. The supernatant was used for the assay of SOD activity and was determined from the quantity of tetrazolium salt formed. One unit of SOD is defined as the amount of enzyme needed to exhibit 50% dismutation of superoxide anion.

#### *5.1.5. Glutathinoe Peroxidase (GPx)*

GPx activity was measured indirectly by a coupled reaction with glutathione reductase. Oxidized glutathione, produced upon reduction of hydroperoxide by GPx is recycled to its reduced state by glutathione reductase and NADPH. The oxidation of NADPH to NADP+ is accompanied by a decrease in absorbance at 340 nm. The rate of decrease in the A340 is directly proportional to the GPx activity in the sample. The tissues from control and exposed mice were homogenized in cold Tris- HCl with 5 mmole/L EDTA and 1 mmole/L dithiothreitol (DTT). The homogenate was centrifuged and the supernatant was used for the enzyme assay. The protocol described in GPx assay kit was followed.

#### *5.1.6. Catalase*

A tissue 5% homogenate of liver or brain was prepared in cold potassium phosphate buffer containing 1mmole/L EDTA. The supernatant that surfaced was used for catalase assay as described in the assay protocol provided by Cayman Chemical Company. The catalase assay kit used the peroxidatic function of CAT for determination of enzyme activity. The enzyme reacts with methanol in the presence of an optimal concentration of H2O2. The formaldehyde produced is measured spectrophotometrically (� = 540 nm) with 4-amino-3-hydrazino-5 mercapto-1,2,4-triazole (Purpald) as the chromogen. Purpald specifically forms a bicyclic heterocycle with aldehydes, which upon oxidation changes from colorless to a purple color. One unit is defined as the amount of enzyme that will cause the formation of 1.0 nmol of formaldehyde per minute at 250°C.

#### *5.1.7. Glutathione S- transferase (GST)*

Tissues from control and experimental mice were homogenized in cold potassium phosphate containing 2 mmole/L EDTA. Homogenates were centrifuged and the supernatants used for the assay of Glutathione S-transferase, GST. GST activity was measured from the quantity of 1-Chloro-2, 4-Dinitrobenzene (CDNB) conjugated to glutathione. The one unit of enzyme conjugates 1.0 nmole of CDNB with reduced glutathione/min/mg.

#### *5.1.8. Statistical treatment of the data*

Data presented in the Tables 4 and 5 are mean ± standard deviation (SD) of 9 animals per group. The data were subjected to analysis of variance (ANOVA) and when means were

significantly different they were further subjected to Tukey test. The percentage variation from controls was calculated and presented in the following results section. A value of P < 0.05 was considered significant.


**Table 4.** The lipid hydroperoxides and antioxidant enzyme activities in the brain tissue. Values are mean *±* Standard deviation (*n* = 9). \*Significant at *p <* 0*.*05, ▪ not significant


**Table 5.** The lipid hydroperoxides and antioxidant enzyme activities in the liver tissue. Values are mean *±* Standard deviation (*n* = 9). \*Significant at *p <* 0*.*05, ▪not significant

#### **6. Results**

The overall short term effect of BDE-209 over a period of four days is a remarkable disruption in the oxidant/antioxidant equilibrium. First, the levels of lipid hydroperoxides in the experimental tissues, liver and brain, have increased considerably. The percent increase in the levels of lipid hydroperoxides in liver was 27.67 % (*p* < 0.05) and in brain was 42.72% (*p* < 0.05) over controls. These results are shown in Table 4 and 5. The activities of SOD showed varied response in brain and liver. In liver, though there was a decrease in activity by 8.03 %, when compared to controls, it was not statistically significant. However in brain there was significant ((*p* < 0.05) increase in activity (38.58%). Another anti-oxidant enzyme activity measured was the GPx. The activity of this enzyme has been reduced in both brain (16.93%) and liver tissues (28.01%). These percentage changes also were significant at *p* < 0.05 for both the tissues.

The enzyme catalase also suffered a similar reduction in activity over a four day exposure to BDE-209. Both in the brain and the liver tissues the catalase activity was lowered. In the brain there was a 24.22% of reduction and in liver it went down by 31.15%. Just like GPx and catalase, GST also can reveal oxidative damage. In the present work GST activities were decreased in exposed liver by 31.3% (*p* < 0.05) and in brain by about 22.73% (*NS)*. All the above results clearly exhibited differential susceptibility of mice liver and brain tissues to BDE-209 – induced oxidative stress.

#### **7. Discussion**

528 Lipid Peroxidation

**6. Results** 

0.05 was considered significant.

significantly different they were further subjected to Tukey test. The percentage variation from controls was calculated and presented in the following results section. A value of P <

Oxidative Stress Marker Control BDE-209 % change LPO 2*.*06 *±* 0*.*63 2*.*94 *±* 1*.*33 +42.72\* SOD 13*.*89 *±* 1*.*77 19*.*25 *±* 2*.*56 +38.58\* GPx 103*.*43 *±* 14*.*77 85*.*92 *±* 5*.*61 -16.93\* Catalase 58*.*78 *±* 14*.*22 44*.*54 *±* 5*.*9 -24.22▪ GST 0*.*22 *±* 0*.*07 0*.*17 *±* 0*.*05 -22.73▪

**Table 4.** The lipid hydroperoxides and antioxidant enzyme activities in the brain tissue. Values are

Oxidative Stress Marker Control BDE-209 % change LPO 22*.*52 *±* 2*.*76 27*.*31 *±* 1*.*98 +21.67\* SOD 33*.*37 *±* 1*.*59 30*.*69 *±* 1*.*77 -8.03▪ GPx 198*.*77 *±* 26*.*87 143*.*78 *±* 7*.*8 -28.01\* Catalase 134*.*75 *±* 18*.*34 92*.*78 *±* 10*.*54 -31.15\* GST 4*.*32 *±* 0*.*9 2*.*97 *±* 0*.*3 -31.3\*

**Table 5.** The lipid hydroperoxides and antioxidant enzyme activities in the liver tissue. Values are

The overall short term effect of BDE-209 over a period of four days is a remarkable disruption in the oxidant/antioxidant equilibrium. First, the levels of lipid hydroperoxides in the experimental tissues, liver and brain, have increased considerably. The percent increase in the levels of lipid hydroperoxides in liver was 27.67 % (*p* < 0.05) and in brain was 42.72% (*p* < 0.05) over controls. These results are shown in Table 4 and 5. The activities of SOD showed varied response in brain and liver. In liver, though there was a decrease in activity by 8.03 %, when compared to controls, it was not statistically significant. However in brain there was significant ((*p* < 0.05) increase in activity (38.58%). Another anti-oxidant enzyme activity measured was the GPx. The activity of this enzyme has been reduced in both brain (16.93%) and liver tissues (28.01%). These percentage changes also were

The enzyme catalase also suffered a similar reduction in activity over a four day exposure to BDE-209. Both in the brain and the liver tissues the catalase activity was lowered. In the brain there was a 24.22% of reduction and in liver it went down by 31.15%. Just like GPx and catalase, GST also can reveal oxidative damage. In the present work GST activities were decreased in exposed liver by 31.3% (*p* < 0.05) and in brain by about 22.73% (*NS)*. All the above results clearly exhibited differential susceptibility of mice liver and brain tissues to

mean *±* Standard deviation (*n* = 9). \*Significant at *p <* 0*.*05, ▪ not significant

mean *±* Standard deviation (*n* = 9). \*Significant at *p <* 0*.*05, ▪not significant

significant at *p* < 0.05 for both the tissues.

BDE-209 – induced oxidative stress.

The results of this study clearly indicate the susceptibility of mice brain and liver tissues to BDE-209 induced damage when exposed to 0.25 mg/kg body weight for four consecutive days. The oxidative stress markers namely, lipid hydroperoxides, were found to increase in both the exposed liver and brain tissues. The increase in the lipid hydroperoxide levels could be due to an immediate perturbation in metabolizing enzymes in liver [54].The degree to which this can happen is yet open for further research. Liver being an important metabolizing organ hydrolyzes PBDEs in its attempt to eliminate them from the body. In this process the metabolites formed might possess greater toxicity than the parent BDE [55], thus making the liver prone to excessive damage. In the present study, BDE-209 exposed brains also showed significant increase in lipid hydroperoxides. Similar observations of increased peroxidation in the brain regions of adult rats exposed to two different concentrations of BDE-99 [42] lend support to this observation. In another study an increased peroxidation was noted in rat hippocampal neurons during BDE-47 exposure [49]. Additionally, there are reports on BDE-99 exposure in rats causing an increased ROS production in the tissues [47,55]. The mechanism responsible for ROS generation during PBDEs is not clear; however, it is suggested that PBDEs are electron acceptors under standard conditions prevailing in biological systems [51]. Reports on PBDE exposure and their involvement in the release of [3H] arachidonic acid, protein kinase-C translocation, and disruption in calcium homeostasis [56,57] could further offer an explanation for the mechanism of ROS formation [49]. Thus the present observation of increased lipid hydroperoxide levels in the liver and brain tissues of mice substantiates higher lipid peroxidation process and oxidative stress following BDE-209 exposure.

The objective of the second part of this study was to examine how antioxidative enzymes responded to BDE-209 exposure in mice tissues. Although lipid hydroperoxides and lipid peroxidation have increased in the tissues, there was no corresponding increase in all the protective antioxidant enzymes studied, namely, SOD, GPx, catalase and GST in BDE-209 exposed mice tissues with the exception of brain SOD. The elevated lipid hydroperoxides under normal conditions should trigger an increase in antioxidant enzyme activity to suit the increased need to quench them. But in the present study there was a decrease in the activities of GPx, GST and catalse in the liver tissue. A decrease in catalase reflects an inability of the liver to remove hydrogen peroxide. As mentioned already, both catalase and GPx show high cooperativity in their action [58]. During the periods of increased hydroperoxide formation catalase activity depends on GPX activity to keep from being inactivated. On account of decreased GPx activity in BDE-209 exposed tissues of mice, the catalase might be inactivated by higher concentration of hydroperoxides. Glutathione-Stransferase is another antioxidant enzyme whose activity was reduced in liver tissue during BDE-209 exposure. BDE-99 exposure in rats showed a similar response of decreased antioxidant enzymes at 0.6 mg/kg bw [42]. In another report on BDE-99, rat liver and kidney showed an upsurge of lipid peroxidation and unstable antioxidant system [55], rendering support to our observations. The overall lowered activities of SOD, GPx and catalase in liver denote that liver tissue is not able to quench increased ROS and oxidative stress. These findings thus show that BDE- 209 brings about an oxidative stress in liver tissue not only through increased lipid hydroperoxide formation but also by reduced antioxidative enzyme protection.

The responses in the brain tissues were different with respect to SOD, catalase, and GST. Unlike in liver, SOD activity in brain was elevated indicating the presence of some degree of protection in the brain tissue against the oxidative damage. Though catalase and GST activities showed reduction (a trend as seen in the liver tissue) the values however, were not statistically significant. This variation in responses in brain compared to liver suggests that brain tissue was able to cope with increased lipid hydroperoxides to some degree, by way of increased SOD activity. The differences in responses in liver and brain tissues could be due to their intrinsic biochemical and functional differences. Brain with its high rate of O2 utilization, high levels of polyunsaturated lipids tends to respond differently [59]. A report on GST of rat brain regions after BDE-99 treatment showed similar response [47]. Further, the dosage used in this study could have resulted in this response in the brain tissue. A study [42] on the adult rat brains exposed to 2 different concentrations of BDE-99 showed contrasting results on TBARS (indicative of oxidative stress) levels. At lower dosage of exposure, the levels of TBARS were found to be reduced and at higher dosage they were increased. There are reports on rats showing decreased antioxidative enzymes in brain tissue during BDE-99 indicating that not all congeners have the same impact on the tissues. Another observation made by the authors (MCV) is BDE-209 is much less toxic compared to BDE-85 [60].

In summary, exposing mice to BDE-209 for four days has brought about an increased lipid hydroperoxide production as seen in the liver and brain tissues. The consequent peroxidation process has significantly impeded the activity of antioxidant enzymes, GPx, GST and catalase, in liver. Brain tissue on the other hand, showed elevated SOD, lowered GPx without significantly reducing catalase and GST activities. The results of this study indicate that BDE-209 congener of PBDEs has grave potential to disrupt the activity of antioxidant enzymes and induce oxidative stress. Additionally, liver seemed to be more susceptible to this toxicant than the brain. These differences in responses in these two tissues could be due to their inherent biochemical and functional differences.

#### **8. Conclusion**

Plasma membrane covers a cell and acts as a barrier between the internal and external environment thereof. It also regulates the movement of molecules across it. Chemically, the plasma membrane is a bi-layer composed of phospholipids and proteins. Membrane lipids form the bulk of the membrane itself and are more abundantly found than the proteins; however, they are smaller in size. Lipids serve as semipermeable barrier allowing only those materials that are lipid soluble and non-polar in nature to freely cross the plasma membrane. The membrane proteins give structural support and also have several functions including serving as transporters, receptors, enzymes and anchors. Carbohydrates which are attached to the membrane proteins serve as identification markers and help in the differentiation of self from non-self. Thus the plasma membrane is a very important structure of the cell and disruption in its structure leads to disruption in cellular function. The membrane lipids are prone to disruption and loss of selective permeability needed for normal metabolism especially through lipid peroxidation by free radicals.

530 Lipid Peroxidation

protection.

much less toxic compared to BDE-85 [60].

**8. Conclusion** 

findings thus show that BDE- 209 brings about an oxidative stress in liver tissue not only through increased lipid hydroperoxide formation but also by reduced antioxidative enzyme

The responses in the brain tissues were different with respect to SOD, catalase, and GST. Unlike in liver, SOD activity in brain was elevated indicating the presence of some degree of protection in the brain tissue against the oxidative damage. Though catalase and GST activities showed reduction (a trend as seen in the liver tissue) the values however, were not statistically significant. This variation in responses in brain compared to liver suggests that brain tissue was able to cope with increased lipid hydroperoxides to some degree, by way of increased SOD activity. The differences in responses in liver and brain tissues could be due to their intrinsic biochemical and functional differences. Brain with its high rate of O2 utilization, high levels of polyunsaturated lipids tends to respond differently [59]. A report on GST of rat brain regions after BDE-99 treatment showed similar response [47]. Further, the dosage used in this study could have resulted in this response in the brain tissue. A study [42] on the adult rat brains exposed to 2 different concentrations of BDE-99 showed contrasting results on TBARS (indicative of oxidative stress) levels. At lower dosage of exposure, the levels of TBARS were found to be reduced and at higher dosage they were increased. There are reports on rats showing decreased antioxidative enzymes in brain tissue during BDE-99 indicating that not all congeners have the same impact on the tissues. Another observation made by the authors (MCV) is BDE-209 is

In summary, exposing mice to BDE-209 for four days has brought about an increased lipid hydroperoxide production as seen in the liver and brain tissues. The consequent peroxidation process has significantly impeded the activity of antioxidant enzymes, GPx, GST and catalase, in liver. Brain tissue on the other hand, showed elevated SOD, lowered GPx without significantly reducing catalase and GST activities. The results of this study indicate that BDE-209 congener of PBDEs has grave potential to disrupt the activity of antioxidant enzymes and induce oxidative stress. Additionally, liver seemed to be more susceptible to this toxicant than the brain. These differences in responses in these two tissues

Plasma membrane covers a cell and acts as a barrier between the internal and external environment thereof. It also regulates the movement of molecules across it. Chemically, the plasma membrane is a bi-layer composed of phospholipids and proteins. Membrane lipids form the bulk of the membrane itself and are more abundantly found than the proteins; however, they are smaller in size. Lipids serve as semipermeable barrier allowing only those materials that are lipid soluble and non-polar in nature to freely cross the plasma membrane. The membrane proteins give structural support and also have several functions including serving as transporters, receptors, enzymes and anchors. Carbohydrates which

could be due to their inherent biochemical and functional differences.

Free radicals are atoms or molecules with one or more unpaired electrons in their outermost shell. Free radicals with oxygen are called reactive oxygen species (ROS). Examples of ROS include hydrogen peroxide, hydroxyl radicals, and superoxide anions. From time to time free radicals are generated as part of aerobic life during normal metabolism and in some instances these radicals are intentionally generated as part of normal signal transduction process. Superoxide anions are generated in cell organelles such as mitochondria and microsomes. These anions can react with biological molecules the most due to their longer half-life. Another most reactive free radical of the cell is hydroxyl radical. This radical is formed by the hemolytic fission of hydrogen peroxide. Free radicals, due to their high reactivity, attack other macromolecules within their vicinity and cause disruption of these molecules. When these free radicals attack proteins they change their biophysics thereby leading to functional disruption. They also earned notoriety in disrupting DNA and gene expression. It is reported that twenty types of alterations are introduced in the DNA by these ROS leading to variations in the gene expression. Last but not the least, ROS also can attack lipids especially the membrane lipids of the cell and cause extensive damage to the cellular function. Free radicals steal electrons from the polyunsaturated fatty acids (PUFAs) and other lipoproteins of the cells and initiate an attack called lipid peroxidation. The peroxidation of lipids occurs in three steps, namely, initiation, propagation and termination. In particular, lipid peroxidation characterized by low density lipoproteins plays an important role in atherosclerosis.

All cells have antioxidants in place to counteract the harmful effects of free radicals. Antioxidants are of two types, chain breaking antioxidants and preventive type. Vitamins such as E and C are examples for chain breaking type as they donate electrons to the existing free radicals and lessen their degree of reactivity to prevent further oxidative damage. The second category of antioxidants (the preventive type) consists of enzymes that scavenge the free radicals before they initiate oxidative attack. Glutathione is an important antioxidant of the cells whose function includes anti-oxidation, strengthening immune system, building DNA, and detoxification of medications and cancer causing compounds. The diverse functions of glutathione are mainly due to the sulfhydryl group in one of its amino acids known as cysteine. Vitamins C, E and provitamin A also play a role in protecting the body against the free radical induced damage. There are many enzymes inside the cells with antioxidant property. The most important antioxidant enzymes present inside the cells are superoxide dismutase (SOD), catalase, glutathione peroxidase, and glutathione Stransferase. SOD catalyzes the dismutation of superoxide anion to O2 and H2O2. GPx catalyzes the reduction of hydroperoxides and serves to protect the cell from the hostile free radicals. Catalase, another antioxidant enzyme, along with GPx, is essential for

removing the hydrogen peroxide formed during oxidation reactions. GST along with glutathione and GPx neutralizes free radicals and lipid hydroperoxides especially at low levels of oxidative stress.

Polybrominated diphenyl ethers (PBDEs) are organic brominated flame retardants that were introduced into the market in 1970s and are added in large quantities to many commercial and household products such as computers, TVs, foam mattresses, carpets, etc., to inhibit ignition and prevent fire accidents. Ever since they were introduced into the market, deaths due to fire accidents abated significantly. Commercially these chemicals are available in three mixtures, pentaBDEs, octaBDEs and decaBDEs. Currently, deca BDE constitutes 97% of the total production of PBDEs worldwide and it is the only BDE which is in use in the US. PBDEs are lipophilic and resistant to chemical and physical degradation. Due to their persistent nature, they have become widespread environmental contaminants. Avoiding exposure to PBDEs is nearly impossible as they are ubiquitously found in air, water and food. Few toxic studies on PBDEs are reported in laboratory animals. There are some reports indicating that PBDEs can induce disruption in spontaneous behavior, reduced habituation, impaired memory and learning in rats exposed during critical developmental period. When the toxic effect of BDE-209 was explored in the mice at 0.25 mg/kg body weight, it became clearly evident that this particular BDE triggers lipid hydroperoxide generation and increased lipid peroxidation process. Additionally, the antioxidant enzymes were diminished in liver, but in brain the levels of SOD increased without any change in catalase and GST. These results show that BDE-209 has the ability to elicit serious oxidative stress in mice with increased lipid peroxidation and decreased antioxidant protection. Research is underway to understand the effects of BDE-209 on the expression of genes and to find out if it are responsible for impairment of cell cycle regulation and causing apoptosis.

## **Author details**

Mary C. Vagula\* and Elisa M. Konieczko *Biology Department, Gannon University, Erie, PA, USA* 

### **Acknowledgement**

The lead author (MCV) thanks Gannon University for granting the award of Faculty Research Grant. Authors also thank Prof. Gustafson and Dr. Masters for financial support and encouragement.

#### **9. References**

[1] Singer S, Nicholson G (1972) The fluid mosaic model of the structure of cell membranes. Science. 175(4023): 720-731.

<sup>\*</sup> Corresponding Author

[2] van Meer, G (2006) Cellular lipidomics. EMBO Journal. 24:3159-3165.

532 Lipid Peroxidation

levels of oxidative stress.

regulation and causing apoptosis.

Science. 175(4023): 720-731.

 and Elisa M. Konieczko *Biology Department, Gannon University, Erie, PA, USA* 

**Author details** 

**Acknowledgement** 

and encouragement.

**9. References** 

Corresponding Author

 \*

Mary C. Vagula\*

removing the hydrogen peroxide formed during oxidation reactions. GST along with glutathione and GPx neutralizes free radicals and lipid hydroperoxides especially at low

Polybrominated diphenyl ethers (PBDEs) are organic brominated flame retardants that were introduced into the market in 1970s and are added in large quantities to many commercial and household products such as computers, TVs, foam mattresses, carpets, etc., to inhibit ignition and prevent fire accidents. Ever since they were introduced into the market, deaths due to fire accidents abated significantly. Commercially these chemicals are available in three mixtures, pentaBDEs, octaBDEs and decaBDEs. Currently, deca BDE constitutes 97% of the total production of PBDEs worldwide and it is the only BDE which is in use in the US. PBDEs are lipophilic and resistant to chemical and physical degradation. Due to their persistent nature, they have become widespread environmental contaminants. Avoiding exposure to PBDEs is nearly impossible as they are ubiquitously found in air, water and food. Few toxic studies on PBDEs are reported in laboratory animals. There are some reports indicating that PBDEs can induce disruption in spontaneous behavior, reduced habituation, impaired memory and learning in rats exposed during critical developmental period. When the toxic effect of BDE-209 was explored in the mice at 0.25 mg/kg body weight, it became clearly evident that this particular BDE triggers lipid hydroperoxide generation and increased lipid peroxidation process. Additionally, the antioxidant enzymes were diminished in liver, but in brain the levels of SOD increased without any change in catalase and GST. These results show that BDE-209 has the ability to elicit serious oxidative stress in mice with increased lipid peroxidation and decreased antioxidant protection. Research is underway to understand the effects of BDE-209 on the expression of genes and to find out if it are responsible for impairment of cell cycle

The lead author (MCV) thanks Gannon University for granting the award of Faculty Research Grant. Authors also thank Prof. Gustafson and Dr. Masters for financial support

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## *Edited by Angel Catala*

The purpose of this book is to concentrate on recent developments on lipid peroxidation. The articles collected in this book are contributions by invited researchers with a long-standing experience in different research areas. We hope that the material presented here is understandable to a broad audience, not only scientists but also people with general background in many different biological sciences. This volume offers you up-to-date, expert reviews of the fast-moving field of Lipid Peroxidation. The book is divided in four mayor sections: 1-Lipid peroxidation: chemical mechanisms, antioxidants, biological implications; 2-Evaluation of lipid peroxidation processes; 3-Lipid peroxidation in vegetables, oils, plants and meats and 4-Lipid peroxidation in health and disease.

Lipid Peroxidation

Lipid Peroxidation

*Edited by Angel Catala*

Photo by selvanegra / iStock