Background Information .

Background Information

2. Background Information

Radiation is detrimental to life. The biological effects of radiation are the end products of

a long series of phenomenon which are set in motion by the passage of radiation through

the medium. Harmful effects of radiation are brought about through chemical changes in

the cell caused by the ionization, excitation and atomic displacement. Severity and

nature of damage and time at which they appear depends on spatial and temporal

distribution ofradiation energy (Goodhead 1988). Radiation damage to biological system

involves either direct or indirect action depending on whether the energy is absorbed by

the tissue biomolecules or by the surrounding water. Since cells consist of about 60-80%

of water, to a greater extent the biological effects are mediated through the action of

radiation on water (reaction 1 ).

. . . 1

Radiolytically formed free radicals and molecular species react with biomolecules

and bring about the changes in structure and function. Thus, free radical processes are

involved in radiation-induced cellular lethality (Redpath 1981).

Importance of ionizing radiation as a therapeutic agent in the treatment of

pathological conditions such as cancer, lupus and indolent ulcer was recognized probably

first time (on January 27, 1896) by G. Gillman of the Hannemann Medical College

(which later become General Medical College) while examining a dermatitis on the back

of left hand of E. H. Grubbe (a physicist at Chicago running a business making

incandescent lamps, Geissler tubes and Crookes tubes), exposed to the excited Crookes

tubes (X-ray tubes) during the testing. Interestingly, the first patient with carcinoma of

left breast was treated by Grubbe himself (on January 29, 1896) using probably one of

these Crookes tubes as a source of X -rays; just one month after the Roentgen's famous

presentation about X-ray discovery before the Physikalisch-Medcinesche Gesellschaft,

zu. Wurzburg on 20th December 1895. However, as mentioned earlier, the hypothesis of

Bergonie and Tribondeau (1959) which could be stated as follows: "the radiosensitivity

of cells is directly proportional to reproductive activity and inversely proportional to

5

Background Information

degree of differentiation" laid the foundation of radiation therapy of cancer. The cancer

cells have greater reproductive activity and are less differentiated; are expected to be

more vulnerable to the detrimental action of ionizing radiation. Since hypoxia has a

dramatic protective effect against ionizing radiation, the presence of hypoxic cells limits

the success of radiation therapy of cancer (Thomlinson and Gray 1955, Brown and

Giaccia 1994). The use of chemical agents, which can differentially protect the

surrounding cells and/or sensitize the hypoxic cells is desirable strategy to improve the

therapeutic index.

It is generally accepted that the DNA and membranes are the critical targets of

radiation action. The chemical agent which can interact with DNA or membranes could

have radiomodifying effect. Such possibility could be exploited to improve the radiation

therapy of cancer. Phenothiazines are a group of drugs which interact with cell

membranes (Seeman 1972) leading to various changes in membrane characteristics

(Hauser et al. 1969, Seeman et al. 1969, Shenoy and Singh 1985). Phenothiazine

derivatives (Figure I) such as Chlorpromazine (CPZ), promethazine (PMZ) and

trimeprazine (TMZ) have been screened for radiomodifying efficiency (Shenoy and

Singh 1985, Luthra and Kale 1995, Kale 1996, Varshney and Kale 1996).

Phenothiazine drugs are shown to sensitize hypoxic E. coli Blr to y-rays at

concentrations varying from 25 to 100 J..IM with enhancement ratios (e.r.) varying from

1.8 to 5.0 (Shenoy et al. 1975, 1976, Maniar and Singh 1983). The combined effect of

CPZ at 0.1 mM and procaine HCl (25 mM) when present during hypoxic irradiation of E.

coli Blr in buffer was found to be identical to the effect of procaine HCl alone (Shenoy et

al. 1976). On the other hand, when procaine HCl was added to E.coli Blr irradiated in the

presence of CPZ under hypoxia, the magnitude of cellular lethality was significantly

increased (e.r.=5.0). This was explained in terms of procaine induced molecular

deformation of cell membrane, leading to the inhibition of its repair or restitution from

radiation damage. This possibility was also suggested by Yatvin (1976) and Yonei

(1979).

A protective effect was also observed in euoxic mammalian cells in vitro as well

as bacterial cells for phenothiazines viz CPZ, PMZ and TMZ. The radioprotective

6

Figure I. Basic structure of phenothiazine molecule

Where,

For CPZ:

PMZ:

TMZ:: R 1=H '

R2= -CHzCHzCHzN(CHJ)z

R2= -CHzCH(CHJ)N(CHJ)z

R2= -CHzCH(CHJ)CHzN(CHJ)z

Background Information

efficacy of these drugs was shown to increase with increasing concentration and then

decrease (Maniar et al. 1984).

The selective toxicity of radiosensitizing drugs towards hypoxic cells is of

considerable interest to the chemotherapists and radiotherapists. CPZ was shown to be

preferentially toxic towards hypoxic E.coli Blr (Shenoy and Singh 1978). The magnitude

of its toxicity under hypoxia was three times greater than oxic conditions. Cells held

under chronic hypoxia in the presence of drug when irradiated subsequently, also under

hypoxia, demonstrated an effect almost twice that of the 'oxygen effect' (Shenoy and

Singh 1978). This enhanced radiation sensitivity was found to increase by raising the

temperature during irradiation (Shenoy and Singh 1985).

The euoxic radioprotection and hypoxic radiosensitization in addition to hypoxic

cytotoxicity by phenothiazines were also have been reported for Chinese hamster cells

(CHO) (Lehnert 1983). Ehrlich ascites tumor cells were also reported to be sensitized by

CPZ (Schorn et al. 1983). Additional studies demonstrated that treatment of cells with

CPZ resulted in marked depletion of both non-protein sulphydryls (Shenoy et al. 1982)

and protein sulphydryls (Schorn et al. 1983). Furthermore, CPZ also caused a blockage of

cell progression at G1/S and G2/M and inhibited repair of X-ray induced potentially

lethal damage. Significant changes in cellular morphology following treatment by CPZ

(Schorn et a/.1983) and procaine (Yau 1979) were reported, which may influence their

radiation response. Chemotherapeutic and radiosensitizing effects of CPZ and other

phenothiazines have been also reported in two transplantable murine solid tumors in vivo,

namely a fibrosarcoma and sarcoma 180A (Shenoy and Singh 1980, George and Singh

1984) as well as in spontaneously occurring mammary adenocarcinoma in CBA mice.

CPZ was, however, ineffective in a lymphosarcoma and in the ascites form of sarcoma

180 (Shenoy et al. 1982 a, Shenoy and Singh 1980).

As mentioned earlier, the hydroxyl radical mediated transient species of these

drugs as well as inhibition of post irradiation synthesis ofDNA and protein; and rejoining

ofDNA single strand breaks (Shenoy et al. 1975, Maniar et al. 1984, Yonei et al. 1984,

Maniar and Singh 1985) were attributed to their radiosensitizing property. On the other

hand, their radioprotective effect was ascribed to the fluidization of membrane,

facilitating mobility of non-protein sulphydryls (NPSH) and allowing efficient chemical

7

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repair of oxygen specific damage sites (Maniar eta/. 1984). However, these mechanisms

can not explain differential protection of euoxic cells and sensitization of tumor cells.

Their dual radiomodifying property need to be understood. Our laboratory has been

engaged to find out mechanistic aspects of radiomodifying effects of phenothiazines

which can explain the simultaneous protection to the normal tissue and sensitization of

tumor cells by these drugs. On the basis of result obtained in our laboratory, we were

partially successful in explaining the dual radio modifying effects of phenothiazines.

Since, it is quite clear that phenothiazine drugs selectively kill hypoxic cells in

tumors and protect surrounding normal tissue but not vice-versa, it is likely that the

hypoxia itself eliminates the protective properties of these drugs and make them

radiosensitizers. If this were true, then hypoxia may not be considered as a limiting factor

in radiation therapy of tumors. Since, the physiology of transformed cells and normal

cells is different, it is possible that the property of phenothiazines as sensitizers is

determined by the changed biochemical environment in the hypoxic cells of tumors (Kale

1996).

In tumor cells, pH is suggested to be acidic (Song et a/. 1980). It is also

noteworthy that many tumor cells contain high levels of ferritin (Cohen et a/. 1984).

Ferritin is store-house for iron, each molecule of which may hold 4500 molecules of iron.

Hypoxia is known to enhance the mobilization of iron (Bezkorovainy 1989). Ionizing

radiation is found to induce the release of iron from proteins, which increases further on

lowering the pH (Kale and Sitasawad 1991, Sitasawad 1992). Thus, on irradiation, the

presence of hypoxia, low pH and high content of ferritin provide a favorable chemical

environment for more release and availability of iron in hypoxic cells than normal.

Ferrous ions (Fe2+) are readily oxidized to a ferric ions (Fe3+), which are normally

the stable state of iron. In the hypoxic regions of tumors, the Fe2+/ Fe3+ redox couple

would be expected to have an increased reduction potential due to decrease concentration

of oxygen with distance from blood capillaries. Thus, the concentration of Fe2+ ions in

tumors, particularly in the hypoxic cells is expected to be higher than in well-oxygenated

normal cells. It is, therefore, quite possible that the differential radiosensitization of

tumor cells and radioprotection of normal cells by phenothiazines is related to the

presence ofFe2+ and Fe3

+ (Varshney and Kale 1990,"Kale 1996). Ifthis were true, then

8

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phenothiazines drugs should sensitize to radiation in the presence ofFe2+ions and protect

in presence ofFeJ+ ions.

We have tested this assumption using rat liver microsomes. The microsomes

were irradiated with y-rays and the extent of lipid peroxidation was measured. The

protective effect ofphenothiazines was diminished in the presence ofFe2+ ions. However,

they inhibited radiation-induced lipid peroxidation in the presence ofFeJ+ions (Varshney

and Kale 1990). Since the peroxidation process requires oxygen and many tumors contain

hypoxic cells, we have examined the assumption using radiation-induced changes in

glyoxalase I activity in mouse spleen and liver.

The protective effect of phenothiazines m radiation-induced changes of

glyoxalase I activity was reversed in the presence ofFe2+ ions. However, phenothiazines

further inhibited the radiation effect in the presence ofFeJ+ ions (Luthra and Kale 1995).

Thus, we found similar results as those in microsomes.

Pharmacological studies have shown that, on administration, phenothiazines are

distributed to various organs, and could interact with iron released from protein as a

result of irradiation. Phenothiazines and their analogues are known to form metal

complexes (Luthra and Kale 1995). The presence of hypoxia, low pH and accumulation

of ferritin might enhance radiation-induced release ofF e2+ ions in tumors, particular I y in

hypoxic cells which in tum augment the radiation effect in the presence of

phenothiazines. On the other hand, in well-oxygenated normal cells iron would

predominantly be in its stable FeJ+ form, which might enhance a protective effect of

phenothiazines. Since, these drugs are known to scavenge free radicals and undergo

hydrogen transfer (Varshney and Kale 1990), they are expected to provide differential

protection even in the absence ofFe3+ ions in normal cells. This aspects is also needs to

be examined.

Regulation of cellular functions by Ca2+ ions has now been well established.

Calmodulin plays an important role in the calcium messenger system (Rasmussen 1986 a,

1986 b). Phenothiazines were shown to be calmodulin antagonists (Weiss et al. 1982,

Roufogalis et al. 1983). It appears that these drugs may exert their action through

calmodulin on Ca2+ dependent biochemical processes. When ghost membranes prepared

from erythrocytes of mice were irradiated with y-rays, the fluidity of membranes

9

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decreased with radiation dose and in presence of calmodulin antagonists (phenothiazines)

it increased. Radiation-induced release of Ca2+ was inhibited by phenothiazines. Activity

of acetylcholine estrase (AchE), a Ca2+ dependent enzyme, was found to decreased after

the irradiation. Presence of phenothiazines diminished the radiation-induced inhibition of

AchE. From these studies, it was suggested that apart from scavenging of free radicals,

phenothiazines perhaps exert their euoxic radioprotective effect through Ca2+ dependent

processes. The Ca2+ dependent process and other biochemical processes are need to be

examined to understand the role of phenothiazines as radioprotector.

It is well established that the physical state of the membrane (fluidity) during

irradiation plays an important role in determining the levels of injury and repair following

the exposure (Yatvin et al. 1984, von Sonntag 1987). The phenothiazine drugs are known

to interact with cell membrane leading to various changes in membrane characteristics

(Seeman 1972, Sheetz and Singer 1974, Paphadijopoulous et al. 1975). Since, the

cytochrome P450 system is located primarily in endoplasmic reticulum, the fluidization

of membranes by phenothiazines might influence its function.

Numerous studies support the idea that the lipid conformation or association in

membranes can influence the process of lipid peroxidation (Patterson and Redpath 1977,

Edwards and Quinn 1982, Raleigh 1988). Phenothiazines have been shown to inhibit

radiation-induced lipid peroxidation (Kale and Sitasawad 1990, Varshney and Kale

1990). Lipid peroxidation and cytochrome P450 are known to be interlinked. It was

important to note that phenothiazines were suggested to be substrate for cytochrome P450

system (Breyer and Schmalzing 1977, Tavoloni and Boyer 1980, Murray and Reidy

1989). Phenothiazines were also shown to induce cytochrome P450 system (Mcintosh

and Topham 1972, Mostafa and Weisburger 1980). These facts are suggestive of close

interaction between phenothiazines and the cytochrome P450 system.

The transition metal ions, particularly ferrous/ferric redox couple have been

suggested to influence the radiomodifying property of phenothiazines (Varshney and

Kale 1990, Luthra and Kale 1995). The reduction and oxygenation of cytochrome P450

while interacting with substrate (RH), electron .donors (e) and molecular oxygen (Oz)

could provide ferrous/ferric redox couple which may have a link with the protective

effect of phenothiazines.

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Since phenothiazines were shown to interact and induce the cytochrome P450

system which provides the ferrous/ferric redox couple, it would be interesting to examine

the effect of ionizing radiation on the cytochrome P450 system and its modulation by

phenothiazines.

Cytochrome P450 system plays very significant role in biological functions. It is

essential for disposition of vast number of drugs and foreign compounds. The system is

also important in metabolism of endogenous compounds such as steroids, fatty

acids and prostaglandins (Guengerich and Shimada 1991, Gonzalez 1992, Wrighton and

Stevens 1992). The cytochrome P450 system frequently prevent the lethal dose of

ingested compounds being accumulated within an organism. Several factors

including radiation, influence the activity of cytochrome P450. Although effects of

radiation on the content, change in substrate binding capacity, kinetics and spectral

property of cytochrome P450 have been reported (Knott and Wills 1973, Yukawa and

Nakazawa 1974, Nakazawa et al. 1976, Bernard and Rocquet 1979, Zajac and

Bernard 1982, Bushma 1988, Khakimov 1989, Naumova 1992), the results were not

in agreements. Most of these studies were centred around the cytochrome P450 per se

and very little was reported on the other components of the cytochrome P450 system.

Moreover, radiation doses used were quite high. Therefore, an attempt has also been

made to examine the radioresponse of different components of cytochrome P450

system. The system could be described briefly as follows.

2.1 Cytochrome P450 system

The cytochrome P450 system consists of mainly NADPH-cytochrome P450 reductase,

NADH-cytochrome bs reductase, cytochrome P450 and cytochrome bs. It is located in

membranes of endoplasmic reticulum and mitochondria (Mangum et al. 1970, Ahmad et

al. 1996). This enzyme system is present in all living organisms (Kargel 1996). A brief

account of various components of cytochrome P450 system is given below.

11

Background Information

2.1.1 Cytochrome P450

Cytochrome P450 is the terminal electron acceptor and is the site where interaction

among electrons, drugs and oxygen takes place (Paine 1981 ). Liver shows highest

concentration of cytochrome P450 (Eugene et al. 1992). It is suggested that cytochrome

P450 is selected during evolution to detoxify the atmospheric oxygen (Nebert 1994).

Cytochrome P450 is tightly bound to membrane and is highly amphiphilic protein (Dean

and Gray 1982, Eugene et al. 1992). Although cytochrome P450 catalyze the oxidation of

a variety of xenobiotic chemicals through monooxygenase reaction, it is also found to

have peroxidase activity in which cytochrome P450 catalyzes substrate hydroxylation

using various hydroperoxides as well as H202 as co substrates (Hrycay and 0 'Brien 1972,

Renneberg et a/.1978, O'Brien and Rahimtula 1980, Aust and Svingen 1982, Cadenas

and Sies 1982, Cavallini et al. 1983, McCarty and White 1983, Kappeli 1986, Crivello

1986, Hollenberg 1992, Thompson et al. 1995, Anari et al. 1995, Segura-Aguilar 1996).

2.1.2 NADPH-cytochrome P450 reductase

NADPH-cytochrome P450 reductase contains one molecule of flavin mononucleotide

(FMN) and one molecule of flavin adenine dinucleotide (FAD) (Iyanagi and Mason

1973). A recently discovered enzyme nitric oxide synthase (Bredt et al. 1991) has

regions-homology to NADPH-cytochrome P450 reductase.

NADPH-cytochrome P450 reductase catalyses electron transfer from NADPH to

cytochrome P450 (Lu and Coon 1968, Alvares and Pratt 1990), cytochrome bs (Enoch

and Strittmatter 1979, Ilan et al. 1981), heme oxygenase (Schacter et al. 1972) and to

fatty acid elongase (Ilan et al. 1981) as well as to non nonphysiological electron acceptors

(Williams and Kamin 1962, Kargel et al. 1996). It has been widely used in subcellular

distribution studies as a marker enzyme for the membranes derived from the endoplasmic

reticulum (ER) (Kargel et al. 1996).

2.1.3 Cytochrome b5

Cytochrome b5 is mainly present in the endoplasmic reticulum in the liver. It is also

found in many other tissues (Mangum 1970, Tamura et al. 1988). Cytochrome bs is

12

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connected with NADH and NADPH dependent electron transfer pathways through

NADH-cytochrome bs reductase and NADPH-cytochrome P450 reductase respectively

(Omura 1978, Oshino 1982, Tamura eta!. 1990, Yamazaki eta!. 1996). Cytochrome b5

has been considered to be an electron donor in cytochrome P450-mediated drug and lipid

metabolism (Keyes and Cinti 1980, Tamura et a/.1988, Yamazaki 1996).

2.1.4 NADH-cytochrome b5 reductase

NADH-cytochrome b5 reductase (EC 1.6.2.2), a FAD containing flavoprotein is a

membrane bound enzyme. Although, it is found in all tissues and organs, the highest

concentration is reported in liver (Tamura et a!. 1987). NADH-cytochrome b5 reductase

transfer the electron from NADH to cytochrome bs, which in tum is involved in a

cytochrome P450 mediated drug as well as lipid metabolism (Keyes and Cinti 1980, Lee

and Kariyar 1986, Tamura eta!. 1988, Yamazaki 1996).

2.1.5 Synergism between NADH and NADPH dependent electron transport system

It is well established that NADH and NADPH-dependent microsomal electron transport

system are closely interlinked (Cohen and Estabrook 1971, Sugiyama et a!. 1980,

Yamazaki et a!. 1996). It is proposed that one of the two electrons required by

cytochrome P450 for hydroxylation reaction is supplied by NADH via cytochrome b5

reductase and cytochrome bs (Hildebrandt and Estabrook 1971, Noshiro and Omura

1978,_ Tamura eta!. 1990, Holmans eta!. 1994, Yamazaki eta!. 1996). Cytochrome b5

can also receive electron from NADPH via NADPH-cytochrome P450 reductase (Lu et

a!. 1974, Kargel eta!. 1996). Electron flow in NADH and NADPH-dependent system is

shown in scheme 1.

13

Background Infonnation

NADPH ~ NADPH-cytochrome P450 reductase ~ Cytochrome P450

~~ NADH ~ NADH-cytochrome bs reductase ~ Cytochrome bs

Scheme 1. Electron flow in NADPH amd NADH dependent electron transport system

2.1.6 Reaction Cycle of cytochrome P450

Reduction and oxygenation of cytochrome P450, while interacting with substrate (RH),

electron from donor molecules and molecular oxygen (02) is shown in scheme 2.

Important steps I events are given below:

• The reaction cycle is initiated through the interaction of cytochrome P450 (Fe3l with

substrate leading to formation of their complex [(RH) Fe3+].

• [(RH) Fe3+] undergoes one electron reduction to form ferrous hemoprotein-substrate

complex [(RH) Fe2+]. The electron required for this reduction is donated by NADPH

via NADPH-cytochrome P450 reductase (Blank et al. 1989).

• The reduced cytochrome P450 in above complex binds with oxygen and results in

formation of oxy-complex [(RH) Fe2+(02)]. This oxy-complex has an ability to

release superoxide anion (Rein et al. 1986).

• The second electron is introduced in the cytochrome P450 reaction cycle is provided

either by cytochrome bs or by NADPH-cytochrome P450 reductase (Bonfils et a!.

1981, Schenk:man 1993 ). The interaction of second electron with oxycomplex leads to

formation ofperoxy complex of cytochrome P450 [(RH) Fe2+(022-)].

• From the peroxycomplex, terminal oxygen is removed in the form ofH20 to generate

an iron-oxo intermediate [(RH) (Fe-Oi+J (Rein eta!. 1986, Rein and Jung 1993).

Apart from this, hydrogen peroxide can split off from this peroxycomplex.

• The hydrogen atom abstraction from the iron-oxo intermediate results into formation

of reactive species [(R)(Fe-OH)J+]. Immediate recombination of these reactive

species produces stable product [(ROH) Fe3+]. This process was designated as

14

e NADH --7

bs-R --7 1/~=:::.~-;;-:_:o··

~/ (7Fe3

•(o,] ---7 ~

e I (RH)Fe'•(o,j H,o, ·oH ·o,H

(RH)(Fe-oi+

(R)(Fe-OH)3+

(RH)Fe2+ J .. ··· .... ·······;;:.:.:'''"'::::::::::::::::.-:~.·-·.· .... :~··············-> (RO H)F e

3+

.... ······ .. ····'! xo NADPH --7 P450-R .... ·········

(RH)Fe3+ xoo

~p3+ RH e

ROH (Substrate)

Scheme 2. Schematic outline of the cytochrome P450 system. Fe3+ (ferric-cytochrome P450) Fe2+ (ferrous-cytochrome P450), P450-R (NADPH-cytochrome P450 reductase) b5-R (NADH-cytochrome b5 reductase), bs (cytochrome bs), e (electron from donor molecule).

Background Information

"oxygen rebound" (Groves and McClusky 1976). Finally, the hydroxylated product

dissociates, and the reaction cycle can restart again.

• Interestingly, in the shunt reaction (denoted by dotted line inside reaction cycle), the

substrate can be hydroxylated immediately by peroxides without interacting with an

electron donating system.

In view of the facts that, cytochrome P450 was shown to have peroxidase activity

(Hrycay and O'Brien 1972, Renneberg et a/.1978, O'Brien and Rahimtula 1980, Aust

and Svingen 1982, Cadenas and Sies 1982, Cavallini et al. 1983, McCarty and White

1983, Kappeli 1986, Crivello 1986, Hollenberg 1992, Thompson et al. 1995, Anari et al.

1995, Segura-Aguilar 1996) and it is known to partially replace catalase in protecting the

cells against oxidative stress (Morichetti et al. 1989), an attempt has been made to

examine whether the radiation-induced activity of cytochrome P450 system and its

modulation by phenothiazines linked with the antioxidant potential of animals, using

DTD, GST, SOD and catalase.

2.2 DT -diaphorase

DT-diaphorase (NAD(P)H:quinone oxidoreductase, EC 1.6.99.2) is a FAD containing

flavoprotein and consists of two identical subunits (Lind et al. 1990). It is widely

distributed in animal kingdom except pigeons. Almost all tissues have DT -diaphorase,

however, richest source is liver (Benson et al. 1980, Schlager and Powis 1990, Belinsky

and Jaiswal1993).

DT -diaphorase is unique, as it displays nonspecific reactivity towards NADH and

NADPH and shows a broad electron acceptor specificity, catalyzing the reduction of

quinones, quinone epoxides, quinoneimines, certain aromatic nitro compounds, aromatic

C-nitroso compounds, azo dyes and hexavalent chromium (Lind et al. 1990, Cadenas et

al. 1992). The most striking feature ofDTD is its ability to catalyze two electron transfer

(Iyanagi and Yamazaki 1970) leading to formation of hydroquinones from quinones

(reaction 2).

Q + NAD(P)H + W ~ QH2 + NAD(Pt . . . 2

15

Background Information

Although, some metabolites generated from DTD catalyzed reaction could be

cytotoxic, this enzyme is also shown to have antioxidant property. Indeed, DID belongs

to a family of phase II detoxification enzymes which includes GST and glutathione

peroxidase with other transferases and reductases (Nebert 1994) known for their function

to divert potentially active electrophiles from damaging interactions with nucleophilic

groups of DNA and in tum protect tissues against carcinogenic and mutagenic

compounds (Talay and Benson 1982, Riley and Workman 1992, Ross et al. 1993,).

DTD prevents the formation of semiquinones from quinones by one electron

reduction and in tum inhibits the generation of free radicals (Lind et al. 1982). DTD also

decreases the electrophilic characters of quinones by aromatization of quinonoid ring,

thereby restricting its participation in arylation reaction leading to avoid cytotoxic effects

(O'Brien 1991).

DTD activity is reported to increase with the activity of other antioxidant enzymes

such as SOD, catalase and glutathione peroxidase (Whitney and Frank 1993, Prestera et

al. 1993). Recently, DTD was shown to protect biological membranes against oxidative

damage (Landi et al. 1997). The antioxidant functions of DTD is attributed to its ability

to maintain membrane bound Coenzyme Q (CoQ) in reduced antioxidant state and

provide protection against free radical damage. It is suggested that DTD has been

selected during evolution to act as CoQ reductase to protect cellular membrane

components from free radical damage (Beyer et a/.1996).

2.3 _ Glutathione-S-transferase

Glutathione-S-transferase (GST) (EC 2.5.1.18) is found in most aerobic microorganisms,

plants and animals; and has selenium independent peroxidase activity using orgamc

peroxides, but can not reduce H20 2 (Batist et al. 1986).

The primary function of enzyme, particularly in higher organisms is generally

considered to be the detoxification of both endogenous and xenobiotic alkylating agents

such as epoxides, a, 13-unsaturated aldehydes and ketones, alkyl and aryl halides and

others. The co-factor for reactions catalysed by this enzyme is the tripeptide glutathione

(GSH) composed of glycine, glutamic acid and cysteine. The importance of GSH in the

16

Background Information

protective mechanism is now well established. Thiols act as protective agents against

electrophiles, radical damage and oxidative stress. GST catalyzes many antioxidant

processes ofthiols (Chaudhary eta!. 1997, Dixon eta!. 1998).

To be a radioprotector chemical I drug should lower the radiation effects. The l radioprotective ability of phenothiazines has been tested using lipid peroxidation,j :I- * xanthine oxidase, lactate dehydrogenase and survival of Swiss albino mice.

-¥ * -1-o p~ 18

2.4 Superoxide Dismutase

Superoxide dismutase (SOD) (EC 1.15.1.1) was isolated by Mann and Keilin (1939) as a

copper containing protein from bovine erythrocytes and called as hemocuprein. Its

antioxidant function of catalyzing the dismutation of superoxide radicals was identified

by McCord and Fridovich (1969). The SOD catalyzes the conversion of 02.- to H202 in

the presence of any substrate that provides protons (Harris 1992, Baez et a!. 1994)

(reaction 3).

o2·_ + o2·- + 2w ~ H202 + o2 ... 3

SOD is present in all oxygen metabolizing cells to provide them with an

endogenous defense against reactions of 02.- generated in aerobic biological system.

Since 0 2·- is one of the several reactive species produced by ionizing radiation, the

potential of SOD to function as a radioprotective agent has been investigated in a number

of experimental system (Das 1998).

2.5 Catalase

Catalase (hydrogen peroxide oxidoreductase, EC 1.11.1.6), a ·heme protein with a single

substrate (H20 2) is ubiquitously distributed in tissues of all species, which was first

isolated from ox liver and later from blood and other sources (Aebi 1984, Harris 1992).

Its maximum activity has been found in liver and erythrocytes. It serves two functions: a)

decomposition of hydrogen peroxide and b) oxidation of hydrogen donor e.g. methanol,

17

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ethanol, formic acid, phenols, with the consumption of one molecules of peroxide

(reaction 4 and 5).

2H202 -----;.. 2H20 + 02

ROOH + AH2 -----;.. H20 + ROH + A

4

5

There are a large number of sources of H202 during an aerobic growth of an

organism. The major damage from H202 arises from highly reactive hydroxyl (HO")

radical, generated in the Fenton reaction between H20 2 and metal ions such as F e2+,

which can react with various components of cells, making it desirable for most aerobic

organism to have catalases or peroxidases to circumvent the damage.

>f- * 1J\OYY1 P-a . I 7

2.6 Lipid peroxidation

Lipid peroxidation is a chain reaction (reactions 6 - 12). Spontaneous oxidation of lipid

molecules by oxygen is called lipid peroxidation. The ground state of polyunsaturated

fatty acids (PUF A) is of singlet multiplicity and their reactions with oxygen are forbidden

since the ground state of oxygen is of triplet multiplicity. The lipid peroxidation reactions

thus involve a mechanism that circumvents the spin barrier. One possibility to overcome

this spin barrier is the reaction via free radicals. In radiolytic systems, free radicals

generated from the water (reaction 6) can attack fatty acid chain of membrane lipid and

result in homolytic dissociation of C-H bond (reaction 7) which leads to removal of spin

restriction and subsequently to initiation of peroxidation. The lipid peroxidation process

involves three distinct stages: (a) initiation, (b) propagation and (c) termination.

Initiation

H20 -----;.. HO", H", e-aq, o·-2

LH + HO" -----;.. L" + H20

Propagation

L" + 02 ~ Loo·

LH + LOO" -----;.. LOOH + L"

18

6

7

8

9

Termination

L" + L" ~

Loo· +Loo· ~ Loo· + v ~

LL

LOOL + 02

LOOL

Background Information

10

11

12

A free radical that have sufficient energy to abstract an allylic hydrogen from

methylene carbon of PUF A, can initiate lipid peroxidation process. HO" (hydroxyl

radical) free radical is considered to be responsible for initiation (reaction 7). The

presence of double bond in fatty acids weakens the C-H bond adjacent to the double bond

and makes hydrogen removal easier. The carbon centered radical (L") reacts rapidly with

molecular oxygen to form LOO" (reaction 8). In subsequent much slower reaction, LOO"

attack another lipid molecules (LH) forming non-radical LOOH while generating new

lipid radical (L") (reaction 9). The later can be converted to LOO" on encounter with

oxygen, closing the self propagating cycle (reaction 8). Thus, once initiated, lipid

peroxidation proceeds to establish a chain reaction with a low energy requirement. In

termination, two free radicals combine to yield a non-radical product to end the chain

reaction (reaction 10- 12) (Patterson and Redpath 1977, Edwards and Quinn 1982, Kale

and Sitasawad 1990).

The membranes, apart from DNA, are considered to be critical targets in radiation

damage and cell death. Lipid peroxidation is an important effect of radiation on cellular

membrane (Leyko and Bartosz 1986), which brings out various changes in structure and

function (Kale and Sitasawad 1990). It is used as determinants of biological damage.

Numerous studies support the idea that lipid conformation or association in the

membrane can influence the process of lipid peroxidation (Patterson and Redpath 1977,

Edwards and Quinn 1982, Raleigh 1988). The membrane active property of

phenothiazines (Shenoy and Singh 1985), conformation or association of lipids m

membranes and lipid peroxidation process suggested to be interlinked (Varshney and

Kale 1996).

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Background Information

2. 7 Xanthine oxidase

Xanthine Oxidase (XO) (EC 1.1.3.22) enzyme participates in the oxidation of a wide

variety of exogenous as well as endogenous (purines) substrates. Although XO was mainly

recognised for its role as the rate limiting enzyme in the nucleic acid degradation through

which all purines are channeled for terminal oxidation, recently it came to limelight due to

its involvement in the generation of oxidising agents such as H202 and reactive oxygen

species. The reactive oxygen species formed by XO are known to be responsible for cellular

damage.

XO is a highly versatile enzyme, widely distributed among species ranging from

bacteria to human and within the various tissues of mammals. In mammals, the liver and

intestine have the highest activity of xanthine oxidoreductase system.

XO primarily exists in vivo predominantly as NAD+ dependent form called xanthine

dehydrogenase (XDH) which can be transformed to an oxygen dependent XO form by a

variety of conditions. The intermediate D/0-Form is also known to be present. The D/0

form may represent the first step of conversion of the native D-Form (XDH) into 0-Form

(XO) in the living cells (Kaminski and Jewezska, 1979).

XO is an important source of oxygen free radicals in the biological system (Parks

and Granger 1986, Jaarsveld et al. 1988). These active oxygen species produced by XO are

involved in cause and complications of many pathological conditions such as post ischemic

reperfusion tissue injury, burn injury (Saez et al. 1984), the adult respiratory distress

syndrome (Grum et al. 1987), lung injury resulting from influenza virus infection (~aike

et al. 1990) and rheumatoid (Blake et al. 1997). Therefore, XO is considered to be

biochemical indicator of cellular damage. Recently we have shown its importance and

usefulness in understanding the radiation-induced damage (Kale and Srivastava 1998,

Srivastava and Kale 1998 a, b, c; Srivastava et al. 1998).

In XO catalysed reactions, oxygen is reduced by one or two electrons producing

reactive oxygen species such as superoxide (02·) or hydrogen peroxide (H202) respectively

(Reaction 13 and 14) (Ames et al., 1981).

Hypoxanthine + 02 + H20 ~ Xanthine + 2W + 02.- 13

Xanthine + 02 + H20 ~ Uric acid + 2W + 0 2·- 14

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Background Information

2.8 Lactate dehydrogenase

Lactate dehydrogenase (LDH) (EC 1.1.1.27) is an oligomeric enzyme and it is reported

to be found in all tissues. LDH catalyzes the reversible conversion of pyruvate to lactate

in the presence of coenzyme NADH (Bergmeyer and Bernt 1974) (reaction 15).

LDH

Pyruvate + NADH + H+ ~ Lactate + NAD+ 000 15

LDH is a cytoplasmic marker enzyme which is well known indicator of damage

induced by several factors including radiation (Wills 1985, Reddy and Lokesh 1996,

Chandra and Kale 1998, Deters et al. 1998, Wahl et al. 1998).

2.9 Survival of mice

The survival of animals is one of the most important biological end point for radiation

protection studies. The radiation protection in terms of survival has a direct relevance to

the radiation therapy of cancer. If phenothiazines have radioprotective ability, the

survival time of irradiated mice can be significantly extended on their administration

prior to irradiation. This aspect is also examined using CPZ.

The ability to protect normal tissue from radiation injury has been a long sought­

after goal in the field of protection from radiation exposure and in the use of radiation to

treat malignant disease. Normal tissues are in intimate association with tumors and in the

path of treatment beam are unavoidably irradiated. A wide range of strategies to protect

the critical normal tissues during radiation therapy are being devised. Application of

chemical protectors is one of them. The development of radiation protectors and

understanding their action remained important not only for their potential to increase the

effectiveness of cancer treatment, but also because radiation protectors provide insight

into the underlying mechanism of radiation cytotoxicity. Also the effect of

radioprotectors or therapeutic agents can be attributed to directly acting chemically based

mechanisms like free radical scavenging, the biological and biochemical processes also

21

Background Information

play a significant role in the mechanism of protective action of these agents. The

cytochrome P450 system and antioxidant enzymes might be closely linked with these

processes. Therefore, an attempt has been made in the present work to examine the effect

ofphenothiazines like CPZ, PMZ and TMZ on the cytochrome P450 system, antioxidant

enzymes and biochemical indicators of cellular damage like lipid peroxidation, xanthine

oxidase and lactate dehydrogenase.

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