26

CHAPTER – 3

REVIEW OF LITERATURE

During World War I, sulphur mustard (mustard) gas was the only blistering

agent in major use. It was recognised by a distinctive odor and had a fairly long

duration of effect under normal conditions. Since then, many mustard compounds

other than sulphur mustard have been developed which are odorless and vary in

duration of effectiveness. Out of these, the nitrogen mustard and sesqui mustard

have attracted some attention. Most blistering agents are insidious in action; there

is little or no pain at the time of exposure except with Lewisite and phosgene

oxime, which cause immediate pain on contact. The symptoms are somewhat

delayed. Phosgene oxime produces a wheal (similar to a bee sting) rather than a

watery blister, which the other blistering agents produce. Blistering agents may be

divided into five categories as given below.

Blistering agent Chemical name NATO code 1. Nitrogen mustard 2. Sulphur mustard - Mustard gas 3. Arsenicals - Lewisite 4. Oximes - Phosgene oxime 5. Others - Sesquimustard

2,2-dichlorotriethylamine 2,2-dichloro-N-methyl-diethylamine (Mechlorethamine) 2,2,2-trichlorotriethylamine 2,2-dichloroethyl sulphide methyldichloroarsine phenyldichloroarsine ethyldichloroarsine [dichloro (2-chlorovinyl) arsine] dichloroformoxime 1,2-bis-(2-chloroethylthio)ethane Bis-[2-(2-chloroethylthio)ethyl]ether L (63%) +HD (37%)

HN-1 HN-2 HN-3 HD MD PD ED L CX Q T HL

27

3.1 History The exact date of the first synthesis of sulphur mustard seems to be

unknown: 1820, 1822 and 1854 have all been quoted by various writers. A

German scientist Despretz recorded the formation of a disagreeable smelling

compound produced by the action of ethylene on sulphur chloride. Niemann and

Guthrie separately synthesized and described the properties of sulphur mustard in

1860 (Niemann, 1860; Guthrie, 1860). Sulphur mustard was first used in the First

World War by Germany against French troops at Ypres, Belgium (1917). Since

then, there has been evidence or allegations of sulphur mustard use in 11

conflicts, including by Italy against Ethiopia in 1936, by Japan against China in

1937, by Poland against Germany in 1939, by Egypt against Yemen from 1963 to

1967, and by Iraq against Iran in the 1980s. In addition, its use was threatened in

the early 1990s during the Persian Gulf War (Papirmeister et al, 1991). The last

military use was in the Iran–Iraq war. Over 100,000 Iranians were injured by

sulphur mustard and one-third is suffering from late effects until today. Historically,

mustard agents are the most widely used type of chemical warfare agent

(Marshall, 1984, Smith et al, 1995).

The nitrogen mustards were the result of an effort to create new chemical

agents before the advent of nerve agents. The nitrogen mustards are ammonia

derivatives. Nitrogen mustards were first synthesised in the 1930s. These

compounds were modifications of sulphur mustard and were found to have greater

systemic toxicity than sulphur mustard (Gilman and Philips, 1946; OSRD, 1946).

There were several reasons for the development of this series of agents. First,

they are more immediately toxic than sulphur mustard, especially to the eyes.

Secondly, they tended to break down the cellulose-based filter elements. Thirdly,

they are effective systemic poisons (Compton, 1987). Nitrogen mustards are

included in Schedule 1 A of the chemical weapon convention, with the three

chemicals HN-1, HN-2 and HN-3, specifically listed. Each of the nitrogen mustards

were investigated as a chemical weapon during World War II. HN-1 and HN-3

28

have both been manufactured on a large scale for weapons purposes, and

weaponisation of HN-3 has been carried out.

During WW II, Louis C. Goodman headed a group that was responsible for

the study of the pharmacological effects of nitrogen mustards. The group,

including Alfred Gilman, Frederick Philips, and Roberta Allen, studied the cytotoxic

properties of nitrogen mustard. Louis C. Goodman and Alfred Gilman are the chief

editors of Pharmacological Basis of Therapeutics book. The group expanded their

work to examine the effect of nitrogen mustard on experimental tumor cells in

mice. It was found, but not published until later, that systemic administration of

nitrogen mustard caused dramatic regression of these mouse tumors. These data

formed the experimental basis of the first clinical trials of nitrogen mustard as a

cancer chemotherapy agent (Gilman, 1946, 1963; Gilman and Philips, 1946;

Rhoads, 1947). The clinical trials with nitrogen mustards were carried out in

several other locations. By 1948, close to 150 patients in the terminal stages of

Hodgkin's disease, lymphosarcoma, or certain leukemias had been treated with

this agent (Gilman and Cattell, 1948). The best results were obtained in cases of

Hodgkin's disease. Derivatives of nitrogen mustard (like cyclophosphamide,

melphelan and ifosphamide) are still used today, particularly for treatment of

various types of cancers due to their strong cytotoxic effects (Goodman and

Gilman, 2006).

None of the nitrogen mustards has been used on the battlefield and none is

included in U.S. stockpiles. However, stockpiled by several countries by the time

of World War II. HN-1 was the first successful ammonia derivative of nitrogen

mustard series synthesised. Its war potentials first came under consideration when

laboratory reports showed, blistering potency similar to sulphur mustard and

lewisite with other characteristics such as respiratory damage, degenerative

changes in the digestive tract, and inhibition of cell mitosis. This compound was

first developed to remove warts. Subsequent studies showed that minute

concentration (typically 0.4 mg in 1 kg distilled water) in direct injection would kill

29

certain malignant growths and tumors. Systemic toxicity occurred among

pharmacologist and laboratory workers.

HN-2 appears to have been formulated about the same time as HN-1.

While HN-1 is a pharmaceutical agent that became a military agent, HN-2 was a

prospective military agent that became a pharmaceutical substance known as

mustine or mechlorethamine. It was found useful for chemotherapy of certain

neoplasm; for many years, it was a mainstay in cancer chemotherapy until it was

replaced by other compounds (Jacobson et al, 1946; Gilman, 1963; Goodman et

al, 1946; Rhoads, 1946). HN-2 molecule is asymmetrical, is unstable upon storage

through polymerisation, forming numerous condensation products. Otherwise this

is a very dangerous compound with high vapour pressure, having an appreciable

vapour density, and being highly volatility. HN-2`s reactivity makes it a good

antineoplastic substance for destroying tumors if injected directly. Since the

human body does not detoxify HN-2, such therapy is a very complicated process.

Although not considered seriously as a military agent by the major powers.

HN-3 was the last of the three nitrogen based blister agents standardised

under an HN code. Whereas HN-1 was designed as a pharmaceutical and

become a military agent, and HN-2 was designed as a military agent and become

a pharmaceutical, HN-3 was designed as a military agent and remained as such.

HN-3 is the principal representative of the nitrogen mustards because its vesicant

properties are almost equal to those of sulphur mustard. It is also most stable

upon storage of the three-nitrogen mustards. The nitrogen mustards do not store

well, due to a tendency to polymerise into quaternary ammonium salt, which are

not reabsorbed into solution. Although HN-3 is the most stable of the series, it too

will decompose in munitions. For this reason HN-3 never superceded distilled

sulphur mustard in NATO inventories. Toxicologically, HN-3 and sulphur mustard

are similar, and also HN-3 has a pronounced lacrimatory effect and rapidly

produces skin rashes. Any nation with pesticide production capacity can produce

the nitrogen mustards.

30

3.2 Structure The CAS Registory number of the mustard agents is given below;

Mustard agents CAS Registory no. HN-1 538-07-8

HN-2 51-75-2

HN-3 555-77-1

Sulphur mustard 505-60-2

The structures of nitrogen mustards and sulphur mustard are shown in

Figure.3.1

HN-1 has a simple structure, in which the hydrogen bonds of ammonia

(NH3) have been replaced with an ethyl group and two chloroethyl groups.

HN-2 is formed around ammonia in which two of the hydrogen bonds are

replaced by chloroethyl (ClC2H4) groups and the third replaced by a methyl (CH3)

group.

In HN-3, the hydrogen bonds of ammonia have been replaced with three

chloroethyl (ClC2H4) groups. HN-3 is the least volatile of the HN series.

The HN series is developed from nitrogeneous compounds. The immediate

parent compounds are triethylamine, (C2H5)3N, and triethanolamine, N(C2H4OH)3.

Sulphur mustard has a simple structure, consisting of two ethyl groups

bound together around an atom of sulphur. Chlorine bonds have replaced the

outer two hydrogen bonds along the central axis.

31

3.3 Physicochemical properties of mustard agents Besides, toxicity mechanisms, the physical properties of the agent itself and

its formulation also present similarly important threat considerations. Selection of

agents and agent formulations can be used to affect differential impacts with

respect to droplet size and liquid deposition, agent persistence, and agent

volatility. The classic chemical warfare agents have a tremendous range of

volatility and volatility can be a determinant in deciding which agents will be used

(US Departments, 1990). Agents such as hydrogen cyanide (1,000,000 mg/m3)

and sarin (22,000 mg/m3) are relatively volatile; they present an immediate, but

short-lived, threat. These agents are referred to as nonpersistent (i.e. they

vaporize rapidly after delivery). Alternatively, agents such as VX (10 mg/m3) and

sulphur mustard (965 mg/m3) tend to fall largely in droplets, with less vaporization,

and remain on exposed surfaces for a long time. These agents are called

persistent. Formulation is also used to manipulate the fate of the agent. Soman,

VX, Lewisite, and sulphur mustard can be mixed with high-molecular-weight

thickeners to increase droplet size and thereby decrease primary vaporization.

Such additives are generally used to promote efficient agent deposition on the

target site. Thickeners can also increase agent persistence and may hamper

decontamination efforts (Sidell et al, 2002). The use of persistent agents such as

mustard and VX may pose a threat for many days. Though such types of data on

vapour pressure and persistency are reported for nerve agents and sulphur

mustard, but for nitrogen mustards, they were not properly analysed. Yet

stockpiled by several countries during World War II, they were not used. The

physicochemical properties of mustard agents are given in Table 3.1 (ATSDR,

1992).

Appearance: - While comparing with pure sulphur mustard, which is a

colorless liquid. HN-1 and HN-3 are colorless to pale yellow oily liquid and

HN-2 is pale amber to yellow oily liquid.

32

Odour: - Distilled technical grade sulphur mustard is nearly odorless to

garlic or mustard odour, while all nitrogen mustards possess a charecterstic

odour. HN-1 has a faint, fishy, or musty odour. HN-2 has fruity odour in high

concentrations and a fishy smell in low concentrations. HN-3 is odorless

when pure but has been reported to have a butter almond odour.

Solubility: - The nitrogen mustards are not very soluble in water. The

reported values are approximately 0.16 g/L for HN-1 and HN-3 and 12 g/L

for HN-2 (Franke, 1982). They are freely soluble in acetone and other

organic solvents like ether, benzene etc. The solubility of HN-3 in water is

less than that of sulphur mustard. HN-3 is soluble in organic solvents viz.

ether, benzene, and acetone. Sulphur mustard is insoluble in water but has

good solubility in organic solvents. Mustard agents will not travel through

groundwater in solution because of its low solubility (Rosenblatt et al,

1975).

Freezing point - It is important to know the freezing point of a chemical

agent, because dissemination characteristics vary markedly with physical

state. For example, sulphur mustard can freeze in a spray tank at low

temperatures and cannot be dispensed. Below their freezing points, most

agents become unreliable in creating casualties by direct effect. The effects

result from secondary transfer. Warming the frozen agent upon entering an

enclosure or area where the temperature is high enough will melt or

vaporise the agent.

Freezing point of sulphur mustard is 14oC; whereas all nitrogen

mustards freeze below 0oC. HN-1 freeze at −34°C, HN-2 freeze at −65°C to

−60°C and HN-3 freeze at −37°C.

Boiling point: - The boiling point represents the highest usable

temperature of a liquid agent. It can be used to estimate the persistency of

a chemical (under a given set of conditions). The reason is that the vapour

33

pressure and the evaporating tendency of a chemical agent vary inversely

with its boiling point. The higher the boiling point, the more slowly a liquid

evaporates at ordinary temperatures. Thus, agents with low boiling points

are normally nonpersistent. Those with high boiling points are persistent.

The boiling point of sulphur mustard is 217oC and evaporates slowly. HN-1

(194°C) and HN-3 (256°C) are near to the boiling point of sulphur mustard.

HN-2 is having a very low boiling point of 75°C and it is least persistant

agent in whole mustard agent’s family. The boiling point also gives an

indication of the practicality for decontamination with hot air.

Vapour density: - Vapour density is the ratio of the density of any gas or

vapour to the density of air, under the same conditions of temperature and

pressure. It is a measure of how heavy the vapour is in relation to the same

volume of air. Vapour density helps in estimating how long an agent will

persist in valleys and depressions. The higher the vapour density, the

longer the vapour will linger in low-lying areas.

Agents with low vapour densities rise. Agent AC (hydrogen cyanide)

is the only militarily significant agent that is lighter than air. Chemical agent

clouds with high vapour densities seek lower ground in much the same way

as water poured on the ground. Agents with high vapour densities tend to

evolve into long coherent streamers. For example, agent sulphur mustard

(5.4) is almost five times as heavy as air. Vapour density of HN-2 is equal to

sulphur mustard whereas HN-1 (5.9) and HN-3 (7.1) having higher vapour

density than sulphur mustard.

Liquid density: - The density of a liquid chemical agent is the weight in

grams of one cubic centimeter of the liquid at a specified temperature.

Liquid density is a measure of an agent’s weight in comparison to water

(density 1.0). This comparison gives the specific gravity. Liquids form

layers, and the one with the greatest density sinks to the bottom. The layer

with the lowest density rises to the top. If an agent is much denser than

34

water, it will tend to sink to the bottom and separate out. An example is

mustardlewisite (liquid density 1.66). If an agent has about the same

density as water, such as the nerve agents, it will tend to mix throughout the

depth of the water. The nerve agents are slightly denser than water.

Therefore, the concentration of nerve agent will tend to increase with depth.

Agents that are less dense than water, such as hydrogen cyanide (liquid

density 0.69), tend to float on water.

Sometimes density is not the only factor that determines how an

agent will distribute in water. For example, if sulphur mustard falls on water,

little globs of it will scatter throughout the depth of the water. The larger

ones tend to coagulate and sink, forming a layer at the bottom. The finer

droplets or mist created by the explosion form a layer on top, one molecule

thick, likes an oil slick. Surface tension holds the layer on the surface. If the

slick is very thin, it will be iridescent, reflecting rainbow like colors. If it is a

little thicker, it may not be noticeable.

Liquid density is of interest in computing the chemical efficiency of a

munition, because we always express toxicities in units of weight. For

example, a munition filled with phosgene (liquid density about 1.4 at 20oC)

will contain twice as much chemical agent by weight as a munition of the

same volume filled with hydrogen cyanide (liquid density about 0.7 at 10oC).

It will also have a much higher chemical efficiency.

When compared to sulphur mustard, the density of all three-nitrogen

mustards is less. HN-3 has approximately less equal density to sulphur

mustard.

HN-1 1.09 g/cm3

HN-2 1.12 g/cm3

HN-3 1.24 g/cm3

Sulphur mustard 1.27 g/cm3

35

Vapour pressure: - Vapour pressure is the pressure exerted by a vapour

when a state of equilibrium exists between the vapour and its liquid (or

solid) state. It is the pressure in a closed space above a substance when no

other gas is present. Vapour pressure varies with temperature, so the

temperature must be stated to determine vapour pressure. At any

temperature, any liquid (or solid) will have some vapour pressure, however

small.

Substances with high vapour pressure evaporate rapidly. Those with

low vapour pressure evaporate slowly. The impact of vapour pressure on

the rate of evaporation makes vapour pressure a very important property in

considering the tactical use and duration of effectiveness of chemical

agents. A potential chemical agent is valuable for employment when it has

a reasonable vapour pressure. One with exceptionally high vapour pressure

is of limited use. It vaporizes and dissipates too quickly. Examples are

arsine and carbon monoxide. On the other hand, mechanical or thermal

means may effectively aerosolize and disseminate solid and liquid agents of

very low vapour pressure. Vapour pressure and volatility are related.

Translated into volatility, vapour pressure is most understandable and

useful.

When compared to sulphur mustard (0.11 mmHg), the vapour

pressure of HN-1 (0.25 mmHg) and HN-2 (0.43 mmHg) are more. HN-3

(0.11 mmHg) has equal vapour pressure to sulphur mustard (Munro et al,

1999)

Volatility: - Volatility is the weight of vapour present in a unit volume of air,

under equilibrium conditions, at a specified temperature. It is a measure of

how much material (agent) evaporates under given conditions. The volatility

depends on vapour pressure. It varies directly with temperature. The

volatility of mustard agents are given below (Munro et al, 1999; Davis,

1947).

36

HN-1 2.29 mg/m3 at 25°C

HN-2 3.60 mg/m3 at 25°C

HN-3 0.12 mg/m3 at 25°C

Sulphur mustard 920 mg/m3 at 25°C

All nitrogen mustards, especially HN-3, are less volatile and more

persistant than sulphur mustard. HN-3 is considered environmentally

persistent, whereas HN-1 and HN-2 are considered moderately persistent.

Because of the low volatility of nitrogen mustards, dangerous concentration

will not be found in the atmosphere. The volatility of sulphur mustard is

more than nitrogen mustards and is capable to form high dangerous

concentration in atmosphere.

Hydrolysis: - The mechanism of hydrolysis is similar for all mustard

agents, with formation of a cyclic intermediate. The hydrolysis in water of

nitrogen mustards is slower than sulphur mustard (Epstein et al, 1973).

HN-1 hydrolyses slowly because of low solubility in water; less

readily hydrolysed than sulphur mustard. HN-2 forms complex condensates

or polymers. HN-2 hydrolyses slowly, except in the presence of alkalis;

products formed are complex polymeric quaternary ammonium salts;

dimerises fairly rapidly in water. HN-3 forms hydrochloric acid and

triethanolamine in dilute solutions; dimer formation in higher concentrations.

HN-3 hydrolyses slowly, except in presence of alkalis; products formed are

complex polymeric quaternary ammonium salts; dimerises rapidly in water.

Sulphur mustard hydrolyses rapidly as compared to nitrogen mustard.

Decomposition Temperature: - The decomposition temperature is that

at which a chemical breaks down into two or more substances. This

temperature can be used to evaluate candidate chemical agents. A low

decomposition temperature (one that is markedly lower than the boiling

point) will usually mean that dissemination of the chemical agent will cause

37

excessive decomposition. All nitrogen mustards and sulphur mustard

decompose before the boiling point is reached.

Stability: - Sulphur mustard is stable in steel or aluminum containers.

When stored in metal container, it shows little corrosive properties. HN-1

polymerizes slowly. So munitions would be effective for several years.

When it stores in metal container and other materials, it is corrosive to

ferrous alloys. HN-1 is obsolete, this being one of the prime reason. HN-2 is

not stable due to its tendency to polymerize under all conditions; HN-3 slow

and steadily undergoes polymerisation. Munitions effective for several

years, with gradual formation of dark amber polymeric quaternary

ammonium salts, which are not resoluble in their parent compound. HN-3 is

stable enough for use as a bomb even under tropical conditions. However,

the agent darkens and deposits a crystalline solid in storage. Hence,

sulphur mustard was preferred over all the nitrogen mustards.

38

Table 3.1 Physical properties of mustard agents.

Property

HN-1

HN-2

HN-3

Sulphur mustard

Description

Colorless to pale yellow oily liquid

Pale amber to yellow oily liquid

Colorless to pale yellow oily liquid

Colorless liquid

Odour

Faint fishy or musty odor

Faint soapy odor at low concentration; fruity odor at high concentrations

Faint butter almond odor

Slight garlic or mustard like odor.

Molecular weight

170.08

156.07

204.54

159.08

Boiling point (760 mm Hg)

194ºC

75ºC 256ºC (decomposes)

217ºC

Freezing point

-34ºC

-65 to -60ºC

-3.7ºC

+14ºC

Vapour pressure (at 25°C)

0.25 mm Hg

0.427 mm Hg

0.0109 mm Hg

0.11 mm Hg

Liquid density (at 25ºC)

1.09 g/mL

1.15 g/mL

1.24 g/mL 1.27 g/mL

Solubility in water

Sparingly soluble

Sparingly soluble

Practically insoluble

Slightly soluble

Samples of nitrogen mustards and sulphur mustard

39

3.4 Toxicological effects of mustard agents

3.4.1 General effects

The nitrogen and sulphur mustard act first as cell irritants and finally as a

cell poison on all tissues. The first symptom usually appears in 4 to 6 hr after

contact (Noyes, 1946). The higher the concentration, the shorter the interval

between the exposure agent and the first symptom. Local action of the mustards

results in conjunctivitis (inflammation of the eyes); erythema (redness of the skin),

which may be followed by blistering or ulceration; and an inflammatory reaction of

nose, throat, trachea, bronchi, and lungs. Injuries produced by mustard heal slowly

and are much more liable for infection than burns of similar intensity produced by

physical means or by other chemicals.

Mustard’s most important metabolic effect, for its acute toxicity, is inhibition

of cellular glycolysis. Disruption of the metabolic pathway is found after exposure

to nearly all vesicant chemicals, usually due to interference with sulfhydryl-rich

hexokinase enzyme systems (Dixon and Needham, 1946). In the case of sulphur

mustard, inhibition of glycolosis is an indirect result to nucleic acid damage and

repair. Initially, sulphur mustard rapidly alkylates the purine bases (guanine and

adenine) of DNA. Activation of endonucleases then leads to removal

(depurination) of alkylated bases, leaving apurinic sites where DNA breaks occur

readily. The need for DNA repair mechanism activates poly (ADP-ribose)

polymerase, an enzyme that rapidly depletes cellular NADP (Papirmeister et al,

1985; Rankin et al, 1980). Depletion of cellular NADP, which begins within 1 h of

exposure and is maximal after 4 h, inhibits glycolysis, leads to release of tissue

proteases, and ultimately results in cell necrosis. These biochemical changes

parallel the development of tissue injury (Dixon and Needham, 1946; Holmstedt,

1963).

Exposure to sulphur mustard causes irritation, edema, vesication, ulceration

and ultimately cell death (Atkins et al, 2000). Direct contact with sulphur mustard

or exposure to its vapour may lead to severe and slow healing lesions (Mol et al,

1991).

40

3.4.2 Effects on eyes

Nitrogen mustards act more quickly on the eyes than does sulphur mustard.

The eyes are very susceptible to low concentrations of nitrogen mustard, while a

high concentration is required to significantly damage the skin or respiratory tract.

The vapours of HN-1 are irritating to the eyes and nasal membranes even in low

concentration. HN-2 affects the eyes in lower doses than do the other mustards. It

produces toxic effects more rapidly than sulphur mustard. HN-2 is highly irritating

to the eyes and throat; in high concentrations it can cause blindness. Majority of

the symptoms of HN-3 are delayed (4 to 6 h) similar to sulphur mustard (Figure

3.2). In some cases eye irritation, tearing, and sensitivity to light (photophobia)

develop immediately. The median incapacitating dose of HN-3 for eyes is 200 mg-

min/m3.

3.4.3 Effects on Skin

Mild vapour exposure may produce no skin lesions. Severe vapour

exposure or exposure to liquid nitrogen mustard will result in redness of the skin,

causing irritation and itching. Later blisters may appear. The skin lesions are

similar to those caused by sulphur mustard. Blistering of the skin may occur after

liquid exposure, severe or persistent exposure, or vapour condensation in sweat.

Usually a rash will develop from liquid contamination within an hour, replaced by

blistering between 6 and 12 h after exposure.

The body does not detoxify HN-2. Therefore, the effects are cumulative.

HN-2 has the greatest blistering power of the nitrogen mustards in vapour form but

is intermediate as a liquid blistering agent. Skin effects are delayed 12 hours or

longer after exposure.

Exposure to sulphur mustard results in the formation of blisters, the

resulting incapacitation can be temporary or permanent (Papirmeister et al,

1984a,b; Somani, 1992) (Figure 3.3). The blistering response may be due to the

release of proteases that may subsequently damage glycoprotein such as lamilin

that mediate dermo-epidermal attachment (Lindsey and Rice, 1995). Inhibition of

41

mitosis and cell death results from the formation of monofunctional and particularly

the bifunctional intra and interstrand cross-links by the interaction of DNA with

sulphur mustard (Fox and Schott, 1980).

3.4.4 Effects on Respiratory Tract

Effects on the respiratory tract are the same as those of sulphur mustard:

irritation of the nose and throat, hoarseness progressing to loss of voice, and a

persistent cough. These effects can progress to fever and labored breathing.

Bronchial pneumonia may appear after 24 hours.

3.4.5 Effects on GIT

Following ingestion or systemic absorption, the nitrogen mustards injure the

intestinal tract. Severe diarrhea, may be bloody, occur. Ingestion of 2 to 6

milligrams causes nausea and vomiting. Nausea and vomiting are common within

the first few hours after nitrogen mustard exposure, the early nausea and vomiting,

which are generally transient and not severe, may be caused by the cholinergic

activity of nitrogen mustard or because of the unpleasant odour (Anslow and

Houck, 1946). Nausea and vomiting that occur later are probably due to the

generalised cytotoxic activity of nitrogen mustard and damage to the mucosa of

the gastrointestinal tract. Diarrhea is not common, and gastrointestinal bleeding

seems to be even less common (Graef et al, 1948).

3.5 Mechanism of Action of Mustard Agents The toxic effects of mustard agent depend on its ability to covalently bind to

other substances. The chlorine atom is displaced from the ethyl group and the

mustard agent is transferred to a reactive sulphonium ion or imonium ion. This ion

can bind to a large number of different biological molecules (Figure 3.4).

Preferably, it binds to nucleophiles such as nitrogen in the base components of

nucleic acids and sulphur in SH-groups in proteins and peptides. Since some

mustard agents contains two or three “reactive groups", it can also form a bridge

between or within molecules. Mustard agent can destroy a large number of

42

different substances in the cell by means of alkylation and thereby influence

numerous processes in living tissue.

The exact mechanisms by which the mustard agents exert their effects are

complicated and not clearly understood. However, the delay in the response of

cells to exposure suggests that the toxicity is not a direct result of the interaction

between the mustard and the target molecule, but involves subsequent cellular

processes (Bernstein et al, 1985). The effects of nitrogen mustard, sulphur

mustard and their derivatives on a number of cellular components and processes

have been studied and various hypotheses have been proposed.

3.5.1 Cross linking of DNA hypothesis

There are several points of evidence which indicate the involvement of

nucleic acids in the mechanism of action of these compounds. For example,

nitrogen and sulphur mustard exert their earliest visible effects on the nucleus,

causing DNA damage.

It is now well established that nitrogen mustards, sulphur mustard and their

derivatives alkylate DNA (Brookes and Lawley, 1960; Hemminki and Kallama,

1986; Price et al, 1968; Ross, 1962; Sack et al, 1978) and RNA (Shooter et al,

1971, 1974a, 1974b).

There are many sites at which alkylation may occur, N-1, N-3 and N-7 in

adenine; N-3, N-7 and O-6 in guanine; N-1, N-3 and O-2 in cytosine and N-3 and

O-4 in thymine (Fig. no. 3.5) (Lawley and Brookes, 1963; Lawley and Shah, 1972;

Lawley and Martin, 1975; Sack et al, 1978; Shooter et al, 1974a). However, in

DNA (Fig. 3.3), nitrogen mustards and sulphur mustard alkylate preferentially at

the guanine N-7 and adenine N-3 (Benson et al, 1988; Brookes and Lawley, 1960;

Ludlum, 1984; 1987; Ludlum et al, 1986; Price et al, 1968; Raikova et al, 1982;

1983), with the guanine N-7 alkylation predominant. The monofunctional mustard

chloroethyl ethyl sulphide (CEES) has also been shown to alkylate guanine O-6

(Ludlum, 1987; Ludlum and Papirmeister, 1986; Ludlum et al, 1986). Crosslinking

of two guanine residues via the N-7 to produce N, N-bis [2-(7-guaninyl)-

43

ethyl]amine or analogous products (Figure 3.6) is also observed (Hemminki and

Kallama (1986). The relative frequencies of these products in DNA exposed to

nitrogen or sulphur mustard have yet to be established (Fox and Scott, 1980;

Whitfield, 1987). Ludlum et al, 1986 found approximately 63% guanine N-7, 11%

adenine N-3 and less than 0.1% guanine 0-6 alkylation after alkylation of DNA by

CEES. However, approximately 16% of the adducts remain unidentified (Ludlum,

1987; Ludlum et al, 1986).

Some alkylating agents are able to esterify the DNA phosphate groups

(Roberts, 1978). However, although it has been proposed that sulphur mustard

should be able to alkylate these groups (Roberts, 1978), no evidence of this

reaction has been found (Hemminki and Kallama, 1986; Ludlum and Papirmeister,

1986).

The bifunctional nature of nitrogen mustards and sulphur mustard results in

the formation of both interstrand and intrastrand crosslinks via the formation of a

diguaninyl derivatives (Flamm et al, 1970; Roberts, 1978). Crosslinks are also

formed between DNA and protein (Dean et al, 1986a, b; Hansson et al, 1987;

Murray and meyn, 1986; Roberts et al, 1968).

The cytotoxic action of nitrogen mustards and sulphur mustard is not

associated with the inhibition of cell growth as measured by RNA and protein

synthesis (Lawley and Brookes, 1965; Roberts et al, 1968). However, the toxic

effects of these compounds are associated with decreased DNA synthesis. The

decreased DNA synthesis is in turn ascribed to the formation of DNA interstrand

crosslinks that prevent the DNA acting as an effective template for DNA replication

(Lawley and Brookes, 1963). Several studies have demonstrated a correlation

between the number of mustard-induced interstrand crosslinks and cytotoxicity

(Garcia et al, 1988; Hansson et al, 1987; Millar et al, 1986; Ramonas et al, 1981).

This hypothesis is supported by the observation that Chinese hamster cells

hypersensitive to bifunctional alkylating agents exhibit decreased ability to remove

DNA crosslinks (Hoy et al, 1985). In addition, the higher toxicity of melphalan than

nitrogen mustard in human melanoma cells under conditions of equal crosslink

44

formation is related to a lower rate of removal of the crosslinks (Hansson et al,

1987). The decreased sensitivity of cells treated in G2 is then ascribed to the

greater chance of the lesions being repaired before the next S-phase, compared to

cells treated at the transition between G1 and S (Murray and Meyn, 1986).

3.5.2 Poly (ADP-ribose) Polymerase Hypothesis

The enzyme poly (ADP-ribose) polymerase (polyADPR-P) is indispensible

for DNA excision repair (Creissen and Shall, 1982; Durkacz et al, 1980; Hayaishi

and Ueda, 1982; Ueda and Haysishi, 1985). DNA damage stimulates poly(ADP)

ribosylatlon and a concomitant decrease in NAD+ (Durkacz et al, 1980; Ueda and

Hayslshi, 1985). Inhibition of Poly (ADP-ribose) Polymerase inhibits the ligation

step (DNA ligase II) in DNA repair (Creissen and Shall, 1982). The NAD+

depletion and cell death induced by the DNA alkylating agent dimethylsulphate are

prevented by Poly (ADP-ribose) Polymerase inhibitors (Stubberfield and Cohen,

1988).

Dean and Fox (1984) and Papirmeister et al (1985) have proposed a

generalised disturbance of cell metabolism involving nucleotide pool imbalance

because of intoxication by mustards. According to Papirmeister et al (1985) the

sequence of events following exposure to mustard is as follows:

i. DNA alkylation;

ii. Spontaneous and enzymatic depurination resulting in apurinic sites and DNA breaks;

iii. Activation of poly(ADP-ribose)polymerase by the accumulation of DNA breaks;

iv. Lowering of cellular NAD+;

v. Inhibition of glycolysis and accumulation of G-6-P;

vi. Stimulation of NADP+-dependent pentose phosphate cycle;

vii. Protease release and

viii. Pathological changes.

45

A number of the steps in the sequence have been demonstrated to occur.

Depurination of alkylated bases is known to occur (Verly, 1980). Treatment of

human skin grafts and leukocytes resulted in a dose-dependent decrease of up to

40% in the NAD levels (Meier et al, 1984; 1987; Papirmeister et al, 1985). More

importantly, in the leukocyte work, the decrease in NAD+ only occurred in those

cells (lymphocytes) containing poly(ADP-ribose)polymerase (Meier et al, 1987).

Finally, inhibitors of poly(ADP-ribose)polymerase inhibited the decrease in NAD+

(Gross et al, 1985; Papirmeister et al, 1985). However, there are some aspects of

the hypothesis which remain to be established. Although some monofunctional

alkylating agents are known to stimulate the synthesis of poly (ADP-ribose)

(Juarez-Salinas et al, 1979), Graham and Fox (1983) did not observe such an

increase in nitrogen mustard treated cells. Secondly, although the NAD+ decrease

could be blocked by inhibitors of poly (ADP-ribose) polymerase or niacin, there

was no report of a concomitant reduction in the cellular damage (Gross et al,

1985; Meier et al, 1984; 1987; Papirmeister et al, 1985). The use of inhibitors of

poly (ADP-ribose) polymerase is also not ideal since these compounds also inhibit

the DNA repair required for the cells to recover (Meier et al, 1984). Also, as

pointed out by Whitfield (1987) other inhibitors of NAD+ synthesis such as

3-acetylpyridine and 6-aminopyridine should also be vesicants.

3.5.3 Oxidative Stress and Glutathione Reduction Hypothesis

DNA is an important but not the only target in the cellular aspect of nitrogen

mustards and sulphur mustard intoxication (Vijayaraghavan et al, 2005). Berger et

al (1979) proposed a general hypothesis for the mechanism of cell death.

Oxidative Stress (OS) is a general term used to describe the steady state level of

oxidative damage in a cell, tissue, or organ, caused by the reactive oxygen

species (ROS). This damage can affect a specific molecule or the entire organism.

Reactive oxygen species, such as free radicals and peroxides, represent a class

of molecules that are derived from the metabolism of oxygen and exist inherently

in all aerobic organisms. There are many different sources by which the reactive

oxygen species are generated. The oxygen species includes O2-. (Superoxide),

46

.HO (Hydroxyl), HO2 (Hydroperoxyl), ROO. (Peroxyl) and RO (alkoxyl) as free

radicals, and H2O2 (Hydrogen peroxide), HOCl (Hypochlorous acid), O3 (ozone)

and 1O2 (singlet oxygen) as nonradicals. The oxidants or free radicals are species

with very short half-life, high reactivity and damaging activity towards

macromolecules like proteins, DNA and Lipids.

The second major hypothesis to explain the effects of mustard agents is

that it reacts with the intracellular free radical scavenger glutathione(GSH), thereby

depleting it, resulting in a rapid inactivation of sulfhydryl groups and the

consequent loss of protection against oxygen-derived free radicals, specifically

those causing lipid peroxidation (Gentilhomme et al, 1992). In 1987, Orrenius and

Nicotera (1987) established that menadione-induced depletion of GSH resulted in

loss of protein thiols and inactivation of sulfhydryl-containing enzymes. Included in

this class of thiol proteins are the calcium and magnesium adenosine

triphosphatases, which regulate calcium homeostasis. With the inactivation of the

enzymes that control thiol proteins, intracellular calcium levels would increase.

High calcium levels within the cell trigger activation of protease, phospholipases,

and endonucleases, which could give rise to the breakdown of membranes,

cytoskeleton, and DNA that, would result in cell death. A report (Ministry of

Defence, U K) suggested that this mechanism could be activated by mustards and

might be the mechanism of mustard injury. While several aspects of the thiol

calcium hypothesis (e.g. release of arachidonic acid and decrease in membrane

fluidity) have been observed in cell cultures following sulphur mustard exposure,

(Ray et al, 1995) no definitive studies have drawn an association between calcium

disruptions and mustard-induced pathology. Another proposed consequence of

the mechanism—based on the depletion of GSH following mustard exposure—is

lipid peroxidation (Miccadei et al, 1988; Paulet, 1952). According to this

hypothesis, depletion of GSH allows the formation of oxygen-derived free radicals.

The oxidizing compounds thus formed will react with membrane phospholipids to

form lipid peroxides that could, in turn, lead to membrane alterations, changes in

membrane fluidity, and eventual breakdown of cellular membranes. As previously

mentioned, studies (Ray et al, 1991) have shown changes in membrane fluidity

47

following sulphur mustard exposure. In addition, in 1989, Elsayed and colleagues

(Elsayed et al, 1989) demonstrated the presence of lipid peroxidation indicators in

the tissue of mice exposed to subcutaneous butyl mustard. However, as with the

thiol–calcium hypothesis, no studies have directly linked lipid peroxidation with the

mustard-induced injury.

Nitrogen mustards like sulphur mustard, alkylate DNA, which leads to

apoptosis and necrosis (Bienvenu et al, 1992). Most of the cellular damage

produced by HN-2 results from the production of free radicals, which directly react

with biological membranes, causing lipid peroxidation and cell damage (O`connor

et al, 1991 and Khan et al, 1992). Second messengers or mediators are generated

from membrane lipid peroxidation or DNA strand breaks, and are potent activators

of the apoptotic signal (Buttke and Sandstrom, 1995). Among the various

processes that may protect cells from oxygen radical–induced apoptosis, the

enzyme glutathione peroxidase appears to be particularly important. This enzyme

catalyzes the reduction of peroxides by glutathione.

Glutathione occurs in high concentration in cells and acts as a redox agent

(Scott and Eagleson, 1988). The Orrenius theory of toxicity suggests that depletion

of glutathione (GSH) is one mechanism by which compounds kill cells (see

Whitfield, 1987). The main function of GSH is indeed to protect tissue and DNA

against the damaging effect of hydroperoxides, and the amount of cellular

glutathione has been found to play a role in the defence against DNA damage

induced by HN-2 (Meister and Anderson, 1983). The involvement of GSH in the

toxicity of nitrogen and sulphur mustard is still unclear. Several groups have

reported increased levels of sulphydryl groups in cells resistant to phenylalanine

mustard (Begleiter et al, 1983; Suzukake et al, 1982). In addition, the formation of

a diglutathione conjugate between phenylalanine mustard and GSH has been

demonstrated (Dulik et al, 1986). Reduction of GSH levels by buthione

sulphoximine or dietary manipulation sensitizes cells, which are resistant to

phenylalanine mustard (Somfal-Relle et al, 1984; Suzukake et al, 1982) while

48

elevation of GSH by diethylmaleate increases resistance (Taylor and Brown,

1988).

Robson et al (1987) found decreased DNA interstrand cross-link formation

accompanied by elevated GSH and elevated glutathione S-transferase activity in

Chinese hamster ovary cells resistant to nitrogen mustard. Conversely, Hansson

et al (1988) observed increased nitrogen mustard toxicity accompanied by

increased DNA interstrand cross-linking in human melanoma cells treated with

nitrogen mustard and D, L-buthionine-S,R-sulfoximine. However, although Raji-

NH2 cells (resistant to nitrogen mustard) have twice the GSH present in sensitive

cells, the reduction of GSH by buthione sulphoximine did not affect the cytotoxicity

(Tan et al, 1987). In addition, Walker 258 cells resistant to nitrogen mustard

exhibited reduced glutathione reductase activity (Dean et al, 1986a,b).

3.5.4 Membrane Effects Hypothesis

Nitrogen mustard is known to have a number of effects on cellular functions

associated with the cell membrane. Treatment of tumor cells or erythrocytes

results in decreased rotational diffusion of membrane components, which is

ascribed to the formation of protein cross-links (Doppler et al, 1985; Grunicke et al,

1985; Levy 1965).

The use of 86Rb+ as a K+ congener has shown that nitrogen mustard

decreases the frusemide sensitive K+ transport, which is mediated by a Na+/K+/CI

cotransporter (Baxter et al, 1982; Doppler et al, 1985; Grunicke et al, 1985;

Wilcock and Hickman, 1988; Wilcock et al, 1988). It is important to note that the

monofunctional mustard (2 chloroethyl)dimethylamine has no such effect (Baxter

et al, 1982; Wilcock et al, 1988). The ouabain sensitive Na+ pump is only affected

at relatively high concentrations of nitrogen mustard (Baxter et al, 1982; Doppler et

al, 1985).

The significance of these observations is unclear. The fact that frusemide

does not affect the cell proliferation indicates that the inhibition of K+ transport by

nitrogen mustard is not sufficient to explain its effects (Doppler et al, 1985;

49

Wilcock et al, 1988). It also remains to be established whether the effect of

nitrogen mustard is direct or indirect (Wilcock et al, 1988).

The trifunctional mustard tris (2-chloroethy!)Amine (HN-3) has been found

to alkylate spectrin and cross-link it to itself and other membrane proteins in

human erythrocytes (Wildenauer and Wegar, 1979). The result of the alkylation is

fixation of the cell shape and protection against metabolically induced chances of

shape (Wildenauer et al, 1980). Spectrin forms the basis of the erythrocyte

membrane skeleton (Bennett, 1985). No reaction between HN-3 and

phospholipids was detected in this experimental system (Wildenauer et al, 1980).

3.5.5 Cytoplasmic Effects Hypothesis

In addition to the effects on nucleic acids and the cell membrane, the

effects on cytoplasmic components have also been studied.

Wildenauer and Weger (1979) and Fung and coworkers (1975) have

observed the alkylation of haemoglobin by nitrogen mustard. In human

erythrocytes the formation of dimers was observed (Wildenauer and Weger,

1979). In isolated sickle cell haemoglobin alkylation at the β2 histidine by nitrogen

mustard was detected (Fung et al, 1975). However, sulphur mustard was not

observed to alkylate haemoglobin (Fung et al, 1975). Sulphur mustard and CEES

have been observed to increase the release of lysosomal enzymes in vitro but only

at extremely high concentrations (Gross et al, 1981). In vivo, treatment of rats with

nitrogen mustard also indicated that these enzymes are unlikely to be involved

early in the response of the cell (Cohen, 1978).

3.6 Medical Management of Mustard Agents

3.6.1 Decontamination Decontamination is the method involving the conversion of toxic chemicals

into harmless products either by degradation or by detoxification. Decontamination

is an important unavoidable part in protection against chemical warfare agents.

The decontaminant should have rapid action, efficiency, non corrosive, safe and

50

washable with water. There are three basic methods of decontamination viz.

physical removal, chemical deactivation, and biological deactivation of the agent.

Though physical and chemical decontamination methods are available to some

extent, biological deactivation has not been developed to the point of being

practical. There is no universal skin decontamination procedure that is effective and

safe. Mustard agents can be hydrolysed by bleach (0.5% hypochlorite solution).

Bleaching powder and chloramines can react violently with sulphur mustard, and

non-poisonous oxidation products are formed. Interestingly, skin burns caused by

mustard exposure do not have the same toxic properties as do thermal burns

(Sollmann, 1957). Skin lesions caused by dermal application of sulphur mustard

can be prevented by adsorbent powders such as Fuller’s earth (a native form of

aluminium silicate), charchol and talcum (Sollman, 1919a,b) as well as household

products such as flour, dutch powder, dry tissue paper and wet tissue paper

removed more than 97% of sulphur mustard (Dacre and Goldman, 1996). The

powder should be used within one minute of exposure. A chemical decontaminant

reported which is effective up to 5 minute post exposure of massive dose of

sulphur mustard (Vijayaraghavan et al, 2002). Wormser et al (2000) developed a

non toxic and nonirritant iodine / povidone-iodine containing preparation which

effectively protect guinea pig skin from 20 minutes post exposure of sulphur

mustard. But there is no recommended decontaminant for nitrogen mustard to

reduce it’s toxicity. There is no specific antidote for nitrogen mustards and sulphur

mustards toxicity. Decontamination of all potentially exposed areas within minutes

after exposure is the only effective means of decreasing tissue damage. Thus, by

the time a patient arrives in the emergency department, decontamination can only

prevent secondary exposure to medical staff; it does not limit the patient’s injury.

Medical treatment is supportive.

51

3.6.2 Drug therapy for mustard agents

3.6.2.1 NAD+ Level Stabilisers

Energy depletion is one of the major consequences of sulphur mustard

toxicity. So, at least in theory, the energy stabilisers have a very important role as

far as medical defence against sulphur mustard is concerned. It was found that

3AB (3-aminobenzamide) and 3 MB ( 3-methoxybenjzmide) prevented the loss of

NAD+ in mouse exposed to sulphur mustard through percutaneous route (Smith et

al, 1991 and Mol et al, 1989). In cultivated human epidermal keratinocytes (Mol et

al, 1989) and in human leukocyte (Meier et al, 1987) the decrease in NAD+ was

reversed by nicotinamide as well as 3- aminobenzamide. But recent studies (Lin et

al, 1994) showed that these compounds did not improve skin lesions significantly

and hence efficacy of these compounds is doubtful.

3.6.2.2 Poly (ADP-ribose) Polymerase Inhibitors

After initial DNA alkylation by sulphur mustard, PARP plays very important

role in the cytopathology induced by sulphur mustard. In addition, it was proposed

that excessive PARP stimulation converts apoptosis to necrosis. It is also involved

in the energy depletion. Thus, PARP inhibitors can restore the energy levels as

well as can change the course of toxicity. Meier and Millard, 1998 showed that two

PARP inhibitors ICD2250 ( 3, 4 - dihydro - 5 - 3- ( methylamino ) - propoxy - 1 - (

2H ) - isoquinolone monohydrochloride and ICD2163 ( 3, 4 - dihydro - 5 -methyl- 1

- (2H) - isoquinolone changed the kinetics of DNA cleavage in human lymphocytes

so that cell death remains apoptotic after sulphur mustard exposure. The

beneficial effect of PARP inhibitors may be related to the blockade of suicide DNA

repair function without affecting the normal functions at cellular and tissue level.

Until date, no work is reported regarding in vivo efficacy of PARP inhibitors.

Niacinamide and 3- aminobenzamide were also shown to be effective in blocking

sulphur mustard induced cell death in human mononuclear leukocytes (Meier,

1996).

52

3.6.2.3 Calcium Channel Blockers and Calmodulin Antagonists

As proposed in thiol - calcium hypothesis, increased calcium may lead to

variety of toxic processes like activation of PARP, phospholipases and proteases.

Calcium channel blockers were tried against dermally exposed mice (Mazumder et

al, 1998) and were found to protect the animals. Calmodulin antagonists such as

trifluoperazine and CGS 9343B were found to prevent biochemical and

pathological changes induced by thermal burns which resembles to burns induced

by sulphur mustard (kim et al, 1996). Further in vivo studies confirmed protective

efficacy of these compounds in water soluble ointment bases against sulphur

mustard induced vesication.

3.6.2.4 Protease Inhibitors

Protease stimulation in the dermo - epidermal junction is directly involved in

blister formation as well as other dermatotoxic effects of sulphur mustard.

Protease inhibitors such as calpain (calcium activated neutral protease inhibitor),

antipain, APMSF and leupeptin inhibited sulphur mustard induced proteolysis in

human peripheral blood lymphocyte culture (Cowan et al, 1992). Niacinamide and

N - acetyl - L - cysteine can also inhibit protease release by inhibiting PARP (Meier

et al, 1987) and by increasing intracellular levels of GSH (Smith et al, 1991)

respectively.

3.6.2.5 Xanthine Oxidase Inhibitors

ATP loss and degradation of ADP increases hypoxanthine levels.

Hypoxanthine is then converted to uric acid by the enzyme xanthine oxidase or it

may be oxidised to reactive oxygen species. By using competitive antagonist of

xanthine oxidase such as hypoxanthine, it is possible to minimise sulphur mustard

induced toxicity (Papiermeister et al, 1991).

3.6.2.6 Anti-inflammatory Agents

One of the initial clinical features of sulphur mustard inhalation is severe

edema and acute inflammatory response, which may lead to cell death (Amir et al,

2000). Sulphur mustard can induce protease release (Cowan et al, 1992),

53

interlukins (Atkins et al, 2000; Ricketts et al, 2000), and various other end products

of cyclo-oxygenase and lipo-oxygenase pathways. Thus anti-inflammatory

compounds may prove beneficial to provide symptomatic relief to a patient

suffering from mild to severe toxic effects of sulphur mustard. Dexamethasone

decreased inflammatory effect of sulphur mustard on alveolar macrophages (Amir

et al, 2000).

3.6.3 Cytoprotection

Cytoprotective agents have been developed as a means of reducing the

toxicity associated with chemotherapeutic agents. The aim of cytoprotective

agents is to improve the therapeutic index of the cytotoxic drugs by reducing the

DLT (Dose Limiting Toxicity) to normal tissue (Links and Lewis, 1999). By

definition, a cytoprotective agent must not compromise the antitumor efficacy of

the chemotherapeutic drug (Links and Lewis, 1999). Ideally, it should be easily

administered, have a reasonable safety profile of its own and cannot interfere with

the delivery of chemotherapy (Capizzi, 1999a; Links and Lewis, 1999). By

improving the patient's tolerance to anticancer therapy, the cytoprotective agents

may offer multiple benefits including potential dose intensification and prolongation

of therapy which may improve response and cure rates. As a result, the costs

associated with supportive care in the event of complications are reduced

significantly and quality of life is improved (Capizzi, 1999a). This also applied to

the toxic effects of nitrogen mustards and sulphur mustard and their prevention by

cytoprotective agents.

The concept of cytoprotection was first introduced by Patt et al (1949) who

reported that rats pretreated with the sulphydryl amino acid cysteine were

protected from Iethal doses of radiation first introduced the concept of

cytoprotection. This sparked the interest in using thiol agents as potential

cytoprotectants (Capizzi, 1999a). Thiol groups contain a nucleophilic sulphur,

which provides an alternative target for the reactive intermediates formed following

treatment with radiation (oxygen free radicals) or alkylating agents (electrophilic

intermediates). These highly reactive species are responsible for both the efficacy

54

and toxicity of anticancer treatments. They bind to various nucleophilic targets

such as DNA, and form covalent bonds without distinction between normal and

cancerous tissue. Therefore, thiol-containing compounds in healthy tissue provide

an alternative nucleophilic target to reactive intermediates generated by radiation

or chemotherapy (Links and Lewis, 1999; Vander and Korst, 1996).

To date, three cytoprotective agents have been approved by international

regulatory agencies. Mesna (Sodium-2-mercaptoethane) is administered to protect

the bladder from toxicity associated with ifosfamide and high-dose

cyclophosphamide whereas dexrazoxane (4-[1-(3,5-dioxopiperazin-1-yl) propan-2-

yl] piperazine-2,6-dione) protects the heart from cumulative cardiotoxic effects

associated with doxorubicin (Capizzi, 1999a). Both of these agents are drug

specific and their protective effects are limited to specific organs. A third agent,

amifostine (WR-2721; Ethyol; S-2 (3-aminopropylamino) ethylphosphoro-thioiç

acid), is the first selective broad-spectrum cytoprotective agent approved by

international regulatory agencies. In this context, broad-spectrum refers to the

protection of multiple organ systems from a broad selection of cytotoxic agents

(Table no. 3.2). The data below shows the cytoprotective affect of amifostine

(Capizzi, 1999a)

The Walter Reed Army Institute of Research originally developed amifostine

as part of an anti-radiation drug- development program of the US Army. Mice,

Dogs and Monkeys that were given Amifostine were protected from lethal doses of

whole body irradiation (Capizzi, 1999a).

Subsequently, it was demonstrated that with Amifostine pretreatment,

normal tissues were protected from the non-specific toxicities of therapeutic doses

of radiation while tumors were not (Yuhas and Storer, 1969). Thereafter, studies

demonstrated that amifostine was effective in protecting normal tissue form the

toxic effects of alkylating agents. cisplatin, anthracyclines and taxanes (Capizzi,

1999c). To date, it is the broad-spectrum cytoprotective agent with the largest

preclinical and clinical database (Capizzi, 1999b).

55

Table 3.2 Cytoprotective spectrum of amifostine (Capizzi, 1999a).

Organs that are affected Drugs

Kidney

Auditory

Peripheral nerves

Bone marrow

Salivary glands

Oral mucosa

Heart

Lung

Esophagus

Small intestine

Colon

Immune system

Testes

Cisplatin

Carboplatin

Cyclophosphamide

Nitrogen mustard

Melphalan

Mitomycin-C

Carmustine

5-flurouracil

ionizing radiation

…others

56

Amifostine is also known to show hepatoprotection in the toxicity caused by

some other toxic compounds, like Dactinomycin that causes extensive liver

damage disease (Valles et al, 1995). How Amifostine act as cytoprotectant is not

very clearly understood, it is being documented that after administration it gets

dephosphorylated to free thiol(WR-1065), which quickly enters the normal tissues

and protects against alkylating agent (Capizzi, 1999c). It can be further oxidized to

disulfide, either symmetrical (WR 33179: may also be cytoprotective (Shigematsu

et al, 1994)) or mixed disulfides with cysteine, Glutathione or Thiol containing

proteins (Figure 3.7) (Capizzi, 1999a). This protection is being attributed to

modulation of glutathione level and protection against lipid peroxidation (Uma devi

and Prasanna, 1990).

The exact mechanism by which WR1065 protects normal tissue has not

been clearly delineated. Several mechanisms have been proposed:

a. The free Thiol can act as a nucleophile that binds directly to and

detoxifies the active spices of alkylating and platinum agents (Capizzi,

1999a; Nici and Calabresi, 1999).

b. WR1065 can act as a scavenger of oxygen free radicals formed after

chemo- or radiation therapy (Capizzi, 1999a; Nici and Calabresi, 1999)

by donating a hydrogen to repair damaged target molecules such as

DNA (Grdina et al, 1999; Nici and Calabresi, 1999).

c. Treating normal cells with WR1065 after treatment has been shown to

reduce radiation- and chemical induced apoptosis. It is hypothesized

that either WR 1065 or WR 3278 (symmetrical disulfide) binds to DNA

and nuclear proteins altering the structures of the internucleosomal

region of the chromatin thus making them Less vulnerable to

degradation. Further work is still required in this area to explore the

Cytoprotective implications of this observation (Capizzi, 1999a).

d. The symmetrical disulfide metabolite of Amifostine, WR33278, also

demonstrates a cytoprotective effect (Grdina et al, 1995). It can bind to

57

DNA and enhance relaxation of DNA super coils that are mediated by

topoisomerase I. It has also been shown that this disulfide metabolite

can protect cells from radiation-induced cytotoxicity and mutations

(Capizzi, 1999a).

e. Recently, selective modulation of modulation of intracellular

glutathione levels by Amifostine, or more specifically, WR1065 has

been proposed as a possible mechanism of action of the

cytoprotective effect. This is hypothesized to occur via enhanced

cysteine delivery into normal cells (Issels and Nagele, 1989; Souid et

al, 1999).

Oxidative stress is one of the important consequences of sulphur mustard

toxicity (Kumar et al, 2001 and Pant et al, 2004). Sulphur mustard has a high

affinity towards glutathione that is one of the important cell constituents.

Antioxidants can protect the normal cell by inhibiting sulphur mustard induced

GSH depletion. Glutathione and its dependent enzymes provide the primary

defense mechanism in cells against the cytotoxic effects of chemotherapeutic

agents (Wolf et al, 1987). The anticancer drugs in which glutathione may play an

important role in detoxification can be divided into two categories:-

i. Drugs that are detoxified by direct conjugation with glutathione.

ii. Drugs that form reactive oxygen species (ROS, which are subsequently

detoxified by the oxidation of GSH to GSSG catalyzed by glutathione

peroxidases (Wolf et al, 1987).

The relative abundance of glutathione in normal and tumor cells is an

important factor in determining the therapeutic index of chemotherapy. Studies

with in vitro and in vivo have showed that the total glutathione concentration is

positively correlated with resistance to chemotherapy and radiation therapy (Bump

and Brown, 1990).

58

The cytotoxicity of mustard agents and production of ROS has been

proposed to result from electrophilic or oxidative stress with depletion of cellular

detoxifying thiol levels including glutathione. Also, ROS are transformed by iron-

requiring reactions into highly toxic oxidants that cause a chain reaction with

membrane phospholipids to form lipid peroxides, leading to loss of membrane

function, membrane fluidity, and finally membrane integrity. Provision and

availability of scavengers of ROS and electrophilic compounds such as

glutathione, sulfhydryls compounds, antioxidants, and substances that will

increase production of endogenous scavengers may be considered protective and

useful. Thereby, the role of cytoprotective compounds such as amifostine and its

analogues like DRDE-07, 09, 10 etc. against nitrogen mustards might be

interesting to be investigated in experimental animal models. Amifostine [S-2(3-

aminopropylamino) ethyl phosphorothioate] and its analogue DRDE-07 [S-2(2-

aminoethylamino) ethyl phenyl sulfide] have been shown to protect sulphur

mustard toxicity (Bhattacharya et al, 2001, and Vijayaraghavan et al, 2001). For

sulphur mustard systemic toxicity a variety of flavonide have been reported to be

very effective (Vijayaraghavan et al, 2006, 2008) but they have been found to be

effective only when the antidote were given as pretreatment and by parenteral

route.

Several antidotes have been reported for the systemic toxicity of nitrogen

mustards and sulphur mustard in experimental animals viz. N-Acetyl cysteine,

Sodium Thiosulphate, Vitamin E (Vojvodic et al, 1985; Khan et al, 1992). All the

antidotes so far screened have given only very limited protection.

Cl CH2 CH2 N CH2 CH2 Cl

C2H5

Cl CH2 CH2 N CH2 CH2 Cl

CH3

Cl CH2 CH2 N CH2 CH2 Cl

CH2 CH2 Cl

Chemical Structures of Mustard Agents

Nitrogen mustard (HN-1) Nitrogen mustard (HN-2)

bis-(2-chloroethyl)ethylamine bis-(2-chloroethyl)methylamine

Nitrogen mustard (HN-3) Sulphur mustard (SM)

tris-(2-chloroethyl)amine 2,2-dichloroethyl sulphide

Figure 3.1 Chemical Structure of Mustard Agents

Cl CH2 CH2 S CH2 CH2 Cl

Figure 3.2 Severe eye lesion followed by sulphur mustard exposure.

Figure 3.3 (A) A typical blister after sulphur mustard exposure.

(B) Extensive skin injury after sulphur mustard exposure.

A

B

Nitrogen mustard/ Sulphur mustard

Reactive intermediates

(carbenium cation)

Figure 3.4 Proposed mechanism of action of mustard agents taken from

Kehe and Szinicz, 2005.

Decreased Cellular Defence

DNA

Damage

Alkylation of Membrane proteins

Reactive oxygen species

Altered protein synthesis

Altered signal transduction

Enzyme inhibition

Alkylation of functional groups

Membrane damage PARP activation

Cell cycle arrest

Metabolic impairment

Necrosis

Apoptosis

Decreased repair Tissue Damage

The Major Purines The Major Pyrimidines

Adenine Thyamine

Guanine Cytosine

Uracil

Figure 3.5 The major purines and pyrimidines shows sites of alkylation.

N

NOH

O

H CH3

123 4

65

6543

2 1

N

N

NH2

OH

N

NOH

O

H12

3 4 56

65

432

1H

H

N

NH

N

N

O

H2N

7

89

65

432

1 7

89

N

N

N

N

H

NH2

H H

(A) 7- ETHYL -THIO ETHYL GUANINE

(B) O-6-ETHYL THIO ETHYL GUANINE

(C) N, N’- BIS [2-(7-GUANINYL)-ETHYL] AMINE

Figure 3.6 Products from alkylation of DNA by nitrogen mustard andsulphur mustard.

CH2CH2NHCH2CH2N

NH

N

NH

H

HN

O

N

NH

N

NH

H

HN

O

N

NH

N

NH

CH2CH2NHCH2CH2ClH

HN

O

N

N

O

H

N

NH

CH2CH2SCH2CH2Cl

H

HN