o ,9*- oo5- 7/70 A LITERATURE REVIEW ON THE MECHANISM OFLn ACTION OF SULPHUR AND NITROGEN. MUSTARDN

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A TIZERATURE REVIEW ON THE MECHANIM OF ACTION

OF SULPHUR AND NITROGEN MUSTARD

Peter J. Gray

MRL Technical ReportMRL-TR-89-24

ABSTRACT

"-The literature describing the mechanism of action of sulphur and nitrogenmustard Wu reviewed. The conclusion drawn is that the available evidencesuggests that DNA is the most importar.t molecular target of thesecompounds. However, the final outcome of poisoning is modulated byother factors such as DNA repair, glutathione levels and drug transport.

ACCOSpi ForNTIS CHA&I

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DI-t o Aj, , f dlor

A-lDist Sýt 1c1di

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Published by DSTO Materials Research LaboratoryCordite Avenue, Maribyrnong, Victoria 3032, Australia.Telephone: (03) 319 3887Fax: (03) 318 4536

* Commonwealth of Australia 1989AR No. 005-717

Approved for public release

AUTHOR

Peter Gray graduated BSc (Hons) (LaTrobe, 1974) and PhD (LaTrobe,1981) in biochemistry. He joined MRL in 1980 and worked on theinhibition of acetyl cholinesterase by nerve agents and the collectionand analysis of electrophyslology data. Recent work has involved3tudy of the effect of nerve agents on neurotranamitter uptake bysynaptosomes.

CONTENTS

Page

1. INTRODUCTION.

2. CHEMISTRY

3. PATHOLOGY

5.1 lUsaue Uptake3.2 Clnicjl Otbseevtions

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4. EXPERIMENTAL OBSERVATIONS

4.1 7usue Explant. - Damage .

4.2 7•Wsu3, axplant. - Inflammatory Response 94.3. Cell Culture and Jolated Cell . 1010

5. MOLECULAR MECHANISMS OF MUSTARD ACTION 125.1 Nucleic Acid,

125.1.1 DNA and RNA alkylation

135.1.2 Consequences of DNA Aikylation 116.1.8- DNA Repair 1414

5.1.3.1 Cross-link repair5.1.3.2 Topolsomermse zi 165.1.3.3 O-6 alkylation repair 165.1.3.4 Poly ADP ribose polymers" 1i

5.1.3.5 Depurination, 17is

5.2 Glutathione5.3 Membrane Effects L85.4 Cytoplasmic Effects 1919

6. PROTECTION

7. DISCUSSION 20

8. REFERENCES 22

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"CBW has comre to be regarded by many peoplo as a dirty andinhumane type of warfare, for which even most professionalmilitary men feel a very deep repugnat.3e. A dispassionateexamination of the facts, however, must surely lead to theconclusion that CBW is a clean, humane form of warfare whepcompared to cther modem weapons systems in current use".

John BemsleyThe Soviet Biochenical Threat to NATOMacmillan Press, 1987

a

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A LnTRATURE REVW ON THE MECHANIM OF ACTION

OF SULPHUR AND N!rO(EN MUSTARD

1. INTRODUCTION

Sulphur mustard, bis(2-chloroethyl)sulphide, (Fig. 1) was first synthesited over a hundredyears ago and first used as a chemical weapon by the Germans on July 12/13 1917 nearYpres in Belgium (Robinson, 1971). This weapon is estimated to ha-ve causedapproximately 400,000 casualties during the First World War (Wachtel, 1941). Since theFirst World War a number of compounds containingthe 2-chloroethyl group have beensynthesized (Robinson, 1971):

.1. 2-chloroethylamines (nitrogen mustards);2. Substituted nitrosamines;3. higher homologuesof sulphur mustard.

The nitrogen mustards (Fig. 2) have found a medical use in the treatment ofHodgkins disease (Calabresi and Parks, 1980). Sulphur mustard has recently been usedextensively by Iraq against Iran (Dunn, 1987). The higher homologues of sulphur mustard(Fig. 3) are reportedly more toxic than their parent corpound (Aleksandrov, 1969).

The seventy years that have elapsed since the first uae of sulphur mustard asa chemical weapon have neither diminished its effectiveness nor led to a clearunderstanding of its mechanism of action. The recent use of sulphur mustard as achemical weapon and the change in attitude to the use of chemical weapons asexemplified by Hemsley (1987) make it imperative that efforts ha made to seekprotective measures against it.

The aim of this paper is to review the literature descrihing the biologicaleffects of the mustardn and mther alkylating agents (Fig. 4) with a view to proposing aprogram of research work to lead to an effective treatment of mustard poisoning.

2. CHEMISTRY

Sulphur mustard and the nitrogen mustards belong to a group of compounds whichspontaneously form cyclic sulfonium or immonium ions in solution (Ross, 1962; Tilley.1989). These reactive species are able to alkylate a number of different cellularcomponents (Ross, 1962).

In aqueous solution, sulphur mustard undergoes nucleophillc substitution by anr 1 mechanism via formation of a cyclic sulfonium cation (Tilley, 1989). The reaction

of the nucleophile with the sulfonium cation is SN2 , however the overall rate iscontrolled by the ionization step and is SN1 (Tilley, 1989). Aliphatic nitrogem mustardsform stable cyclic immonium ions which react with nucleophiles by a bimolecular (S2)mechanism (Ross, 1962).

More extensive discussions of the chemistry of the mustard may be found inRoss (1962) and Tilley (1980).

3. PATHOLOGY

.1 Tissue Uptak.

For the purpose of this review it is sufficient to note that exposure of the skin to sulphurmustard for a few minutes is sufficient for vesication to occur although the appearanceof damage may be delayed for severai'hours (Whitfield, 1987). The fixation andexcretion of the mustards have been reviewed recently by Whitfield (1987) and will notbe considered further.

&2 aiaiWOI Obssmriatk

In humans there is a latent period between exposure to either liquid or vpowur and theonset of symptoms (Sinclair, 1945). Contamination of the skin causes erythema andblistering. Systemic effects such as nausea and vomiting may also occur (Aasted et al,'.1987; Sinclair, 1945). Damage to the eyes, respiratory system and gastrointestinal tract.producing symptoms which may persist for years is also poeqible (Aasted et al, 1987;Colardyn et al, 1986). Severely poisoned individuals exhibit bone marrow depression andmay die from pulmonary infection (Colardyn et al, 1986; Dunn, 1987; Robinson, 1971;Vedder, 1925). Willems (1989) has presnted an extensive clinical study of 170 sulphurmustard casualties evacuated to European hospitals from Iran hetween 1984 and 1986.

Similar inflammatory responses are observed in experimental animals,however, the skin does not form blisters (Bruckert, 1987; Manthel at a&, 1981;Vogt et al. 1984).

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4. EXPERIMENTAL OBSERVATIONS

4.1 Explaats - Damage

The fur- ring animals commonly used for laboratory studies differ from humane intheir res nse to mustard. The damage is greater, occurs more rapidly and does notinvolve blister formation characteristic of human skin (Vogt at al, 1984). In order tostudy the effects of mustard on human skin, Papirmeister et al (1984 a;b) used humanskin gmaf onto congenitally athymic nude mice.

Papirmeister at al (1984a) exposed the grafts to both liquid and vapour andobserved e following:

1. a d dependent latent period between exposure and the onset of any

o r.vable changes;

2. e ema followed by oedema;

3. he orrhage (small);

4. ulc tion;

5. se ration of the epidermis from the dermis just above the basementm brane, and

6. b formation.

Although visible blisters did not form, the response of the grafted tissue wassufficie tly similar to the response of normal human tissue to suggest that thesubsequ nt microscopic observations are relevant to human exposure.

Papirmeister et al (1984a;b) found evidence that damage begins in the basalcell lay r. In addition, the earliest sign of damage appears in the nucleus. In summary,the ligh microscopic observations were (Papirmeister et al, 1984a);

1. m imal changes within the latency period;

2. a arance of pyknotic and karyorrhetic nuclei in the basal cells;

3. in sion of polymorphonuclear leukocytes;

4. a pearance of gape between the epidermis and dermis;

5. croabcess formation;

6. n crosis, and

7. ficroblister formation.

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Electromicroscopic studies of the exposed grafts (Papirmeister et si, 1984b)indicated the following sequence of changes within the basal keratinocytes:

1. condensation and margination of heterochromatin and loss of euchromath;

2. blabbing of the nuclear membrane;

S. appearance of perinuclear vacuoles;

4. swelling of the endoplasmic reticulum;

5. progressive dissociation of rosettes of free polyribosomes;

6. formation of cytoplasmic vacuoles;

7. loe of the integrity of the basal cell plasma membrane;

A. leakage of cell contents and deb-is into the lanuina lucida;

.9. disruption of the anchoring filaments of hemidesmosomes with separatio of theplasma membrane from the lamina densa and accumulation of oedema fluid in thelamina lucida, and

10. infiltration of phagocytes into areas of major damage.

The main criticism of this model system, the tack of visible blistering, wasascribed to failure of the microblisters to coalesce (Papirmeister et al, 1984a). Inaddition, the grafts were lass sensitive. by'a factor of two, than human skin in sittu andhealed much more quickly (Papirmeister et al, 1984a).

4.2 Tim eplants - Inflammatory respoese

Although-rodent and human skins differ in structure and do not show the same type ofblisters, the sequence of events observed following exposure of rodent skin is sufficientlysimilar to human skin to make it a reasonable model (Dannenberg et al, 1985; Vogt et al,1984).

Dannenberg et al (1985) and Harada et al (1985;1987) heve studied theinflammatory respcnse to sulphur mustard injury using explants of rabbit skin containingboth developing and healing lesions. Darnenberg et al (1984) have proposed'tpolymorphonuclear leukocytes accumulate in response to leukokinins released by deadcells and contaminating bacteria. Serum proteins, particularly proteinase 'inhibitors,enter the inflammatory lesion from the circulation and pcssibly play a major role inmodulating the inflammatory response (Harada et al, 1985;1987).

4.8 Oell Culture and Isolated Cells

Nitrogen mustard is transported into cells by a carrier-mediated process, possibly by thecholine carrier (Ritter et at, 1987; Goldenberg et al, 1970;1973). The chloroethyl groupis also thought to bind to the cell surface (Linford et al, 1969).

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Cultured cells exhibit many of the responses observed in tissue explants.Chromosomal aberrations develop and giant cells appear (Bernstein et al, 1987; Hartley-

.Asp and Hyldig-Nielsen, 1984; Roberts et al, 1968). In keratinocyte culturespreferential destruction of the basal cells occurs after exposure to sulphur mustard(Bernstein, et al; 1987). Mouse neuroblastoma cells show induction of axon growth andacetylcholinesterase, consistent with the inhibition of cell division (Lanks et al, 1975;Turnbull et al, 1973).

Treatment of cells with 35S labelled sulphur mustai, :!monstrates alkylationof DNA, RNA and protein (Roberts et al, 1968). DNA synthesis is dec-eased in a dosedependent fashion (Bernstein et al, 1987; Carr and Fox, 1982; Dean and Fox, 1984;.Roberts, 1978; Roberts et al, 1968). In surviving cells, inhibition of RNA and proteinsynthesis is either not observed (Crathorn and Roberts, 1966; Lawley and Brookes,1965; Roberts et al, 1968) or observed on!y at high concentrations of mustard (Vaug-hanet al, 1988). However, both RNA and protein synthesis are diminished in mitochondria(Rutman et al, 1977).

Studies using synchronized cell cultures have shown alterations in theprogression of cells through the cell cycle (Graham and Fox, 1983; Roberts. 1978) andvariations in the sensitivity of the cells throughout the cell cycle. Cells are mostsensitive if treated during G1 (Fig. 5) and least sensitive if treated during mid to late Sphase (Clarkson and Mitchell, 19131; Murray and Meyn, 1986; Roberts et al, 1968). Theterm sensitivity refers to DNA synthesis and cytotoxicity, not alkylation which isunaffected by the position of the cell in the cell c3cle (Murray and Meyn, 1986; Robertset al, 1968). Cells treated with nitrogen mustard may be delayed in G2 after completingthe S phase (Lau and Pardee, 1932).

the The decrease in the rate of DNA synthesis is real, rather than a function ofthe disruption of cell cycle progression preventing cells from entering S phase, and is dueto decreased replicon initiation and decreased DNA chain elongation in preinitiated DNAreplication (Carr and Fox, 1982; Graham and Fox, 1983). The mitotic delify is then aconsequence of the decrease in the rate of DNA synthesis (Roberts, 1978). Theobservation by Roberts et al (1968) that thymidine kinase and DNA polynerase are notaffected by sulphur mustard suggests that the decrease in DNA synthesis is not due toinactivation of enzymes.

The alkylating agents vary greatly in their effects on cultured cells. Half-sulphur mustard is less toxi-, than sulphur mustard (Roberts et al. 1968). Themonofunctional methylmethanesulphonate (MMS) is considerably less toxic than nitrogenmustard (Graham and lox, 1983). According to Roberts (1978) re half-sulphur mustardand 100 MMS alkylations are equivalent to one sulphur mustard alkylation. Themonofunctional agents do not produce the same effects on DNA synthesis elicited by thebifunctional agents (Roberts, 1978; Roberts et al, 1968). The effects of the

* monofunctional agents also appear to be delayed for an additional cell cycle (Roberts,1978). In addition, MMS does not produce the enhanced depression of DNA synthesis insensitive cells, compared to resistant cells, produced by sulphur mustard and methylenedimdthanesulphonate - both bifunctional alkylating agents (Carr and Fox, 1982).

In addition to the decrease in DNA synthesis, there is further evidence of

interference in DNA metabolism. Single strand breaks have been observed in DNAextracted from cells ttreated with sulphur mustard (Bernstein et al, 1985;1987) andchromosomal aberr'tions and sister chromatid exchanges have been observed in cellstreated with both silphur and nitrogen mustard (Fox and Scott, 1980; Hartley-Asp and

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Hyldig-Nielsen, 1984). Finally, both nitrogen and sulphur mustard are mutagenic,teratogenic and carcinogenic in rodents and sulphur mustard is carcinogenic in humans(Fox and Scott, 1980).

The use of culture. cells as experimental models must be treated with somecaution. Different cell lines differ dramati,:ally in their response to alkylating agents.For example, HeLa and Chinese Hamster V79-379A cells require greatl y differentconcentrations of MMS, N-methyl-N-nitrosorea (MNU) and N-methyl-N-nitro-N-nitroioguanidine (MNNG) to achieve the same degree of DNA alkylation (Roberts,19ri8). Murine embryonic fibroblqsts and human melanoma cells also differ in the repairof DNA alkylated by nitrogen mustard (Hansson et al, 1987). Cultured cells may alsochange with continued passage (Erickson et al, 1980; Sognier and Hittelman, 1983).However, although the results obtained using a single cell line must be viewed withca.ation, comparison of strains and the correlati&n of the differe'nces in the response tothe mustards with other metabolic differences may yield valuable information about themechanism of action of these compounds. Indeed, human and rodent keratinocytecultures are becoming an important model system (Bernte'n et al, 1985a;1985b).

In summary, studies using cultured cells lead to the following conclusions:

1. The cytotoxicity of the alkylating agents appears to be due to the inhibition ofDNA synthesis, rather than the inhibition of cell growth;

2. There are differences in the mode of action of bifunctional and monofunctionalalkylating agents and it is generally accepted that bifunctional agcnts are moretoxic than monofunctional agents;

3. DNA damage is observed in cells treated with alkylating agents.

5, MOLECULAR MECHANISMS OF MUSTARD ACTION

The exact mechanisms by which the mustards exert their effects are not clear.However, the delay in the response of cells to exposure suggests that the toxicity is not adirect result of the interaction between the mustard and the target molecule, butinvolves subsequent cellular processes (Benistein et al. 1985). The effects of sulphur andnitrogen mustard and their derivatives on a number of cellular components and processeshave been studied.

5.1 Nucleic Acids

There are several points of evidence which indicate the involvement of nucleic acids inthe mechanism of acticn of these compounds. For example, nitrogen and sulphurmustard e :ert their earliest visible effects on the nucleus, are mutagenic, able toinactivate transforming DNA and cause DNA damage.

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&.1. DNA =md RNA allkyatign

It "s now well established that sulphur and nitrogen mustard and their derivatives alkylateboth DNA (Brookes and Lawley, 1960; Hemminki and Kallama, 1986; Price et al, 1968;Ross, 1962; Sack et al, 1978) and RNA (Shooter et al, 19'i1, 1974a, 1974b).

"There are many sites at which alkylation may occur, 1-1, N-3 and N-7 inadenine; N-3, N-7 and 0-6 in guanine; N-1, N-3 and 0-2 in cytosine and N-3 and 0-4 inthymine (Fig. 6) (Lawley and Brookes, 1963; Lawley and Shah, 1972; Lawley et al,1975; Sack et al, 1978; Shooter et al, 1974a). However, in DNA (Fig. 7), sulphur andnitrogen mustard alkylate prefere,..ially at the guanine N-? and adenine N-3 (Benson etal, 1988; Brookes and Lawley, 1960; Ludlum, 1984;1987; Ludlum et al, 1986; Price etal, 1968; Raikova et al, 1982;1983), with the guanine N-7 alkylation predominant. Themonofunctional mustard chloroethyl ethyl sulphide (CEES) has also been shown toalkylate guanine 0-6 (Ludlum, 1987; Ludlum and Papirmeister. 1986; Ludlum et al,1986). Crosslinkting of two guanine residues via the N-7 to produce N,N-bis 12-(7-guaninyl)ethyllamine or analogous products (Fig. 7) is also observed (Hemminki andKallama, 1986).

The relative frequen .es of these products in DNA exposed to sulphur ornitrogen mustard nave 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 lessthan 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).

Alkylation is strongest in the interiors of sequences of contiguous guanines(Grunberg and Haseltine, 1980; Kohn et al, 1987;1988; Mattes et al, 1986; Shepherd andSage, 1986). This preference is thought to be due to the strongly electronegative.electrostatic potential which results from the clustering of G residues (Mattes et al,1986; Pullman and Pullman, 198i). The sites responsible for interstrand cross links,5*-GC-3, are some of the least reactive sites (Mattes 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 beable to alkylate these groups (Roberts, 1978), no evidence of this reaction has been found(Hemminki and Kallama, 1986; Ludlum and Papirmeister, 1986).

In addition to the electrostatic potential mentioned above, the sensitivity ofthe alkylating agent to the strength of 'he nucleophile, as expressed in the Swain-Scottconstant is also important in determining the result of the alkylstion (Roberts, 1978;Whitfield, 1987). The location of the groups within the DNA structure may also beimportant. For example, the guanine 0-6 and N-7 are in the DNA major groove whilethe adenine N-3 is in the minor groove (Roberts, 1978).

5.1.2 Coemequences of DNA Alkylatiom

The bifunctional nature of sulphur and nitrogen mustard results in the formation of both&nterstrand and intrastrand crosslinks via the formation of a diguaninyl derivative(Flamm et al, 1970; Roberts, 1978). Crosslinks are also formed between DNA andprotein (Dean et al, 1986; Hansson et al, 1987; Murray and Meyn, 1986; Roberts et al,1968). In cultured cells, DNA interstrand crosslinks comprise 30-40% of the total DNA

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crosslinks (excluding intrastrand crosalinks), while the remaining 80-70% is accounted forby DNA-protein croeslinks (Hanason et al, 1987). The major diguaninyl product resultsfrom the formation of intrastrand croeslinks and is estimated to be approximately 60% ofthe total diguaninyl product (Roberts, 1978). This in consistent with the low reactivitycf the 5 -GC-3 site (see Section 5.1.1). Overall, the diguaninyl adducts account forapproximately 16% of the total alkylations (Papirmeister et &l, 1985). The rate of bothinduction and repair of the mnteratrand croselinks is constant throughout the cell cycle(Clarkson and Mitchell, 1981; Murray and Meyn, 1986).

The cytotoxic action of sulphur and nitrogen mustard is not associated withthe inhibition of cell growth as measured by RNA and protein synthesis (IAwley andBrookes, 1965; Roberts et al, 1968).- However, the toxic effects of these compounds areassociated with decreased DNA synthesis (see Section 4.3). The diucrenssd DNAsynthesis is in turn ascribed to the formation of DNA interstrand crobalinks whinhprevent the DNA acting as an effective template for DNA replication (Lawley andBrookes, 19(1. Several stLdies have demonstrated a correlation between the number ofmustard-induced interstrand croaslinks and cytotoxicity (Garcia et al, 1988;Hanneon et al, 1987; Millar et al, 1986; Ramonas et al, 1981). This hypothesis issupported by the observation that Chinese hamster cells hypersensitive to bifunctionalalkylating agents exhibit decreased ability to remove DNA crosslinks (Hoy at al, 1985).In addition, the higher toxicity of melphalan than nitrogen mustard in human melanomacells under conditions of equal crosslink formation is related to a lower rate of removalof the croslinks (Hansson et al, 1987). The decreased sensitivity of cells treated in G2is then ascribed to the greater chance of the lesions being repaired before the next Sphase, compared to cells treated at the transition between GI and S (Murray and Meyn,1986).

However. the hypothesis is by no means proven. In at lest one Cell line,Walker 256 rat mammary carcinoma, there is no simple correlation between cytotoxicityand cross-linking (Dean et al, 1986). In addition, the development of resistance to onebifunctional alkylating agent does not necessarily confer on the cell resistance to others(Aids and Bodell, 1987; Tan et al, 1987; Tew and Wang, 1981). Finally, sensitive andresistant cells do not necessarily differ in their ability to repair nitrogen mustard inducedcrosslinks (Dean et al, 1986; Tan et al, 1987). These observations do not indicate thatthe interstrand croslink is not a lethal lesion, but- do suggest that other factors areinvolved in cytotoxicity. The involvement of other factors is also suggested by the workof Tan et al (1987) who observed decreased crosslink formation in cells resistant tonitrogen mustard, but no difference in the ability of resistant and nonresistant cells torepair croeslinks.

In addition to the formation of DNA-DNA, DNA-protein and RNA-proteincrosslinks, monofunctional alkylation at guanine N-7, guanine 0-6 and adenine N-3 occurs(Section 5.1.1). Alkylation at guanine N-7 accounts for about 60% of the total alkylationand adenine N-3 about 16% (Papirmeister et al, 1985).

The formation of N-3 alkyladenine sensitizes the DNA to endonuclease action(Dorsey et al, 1972). N-7 alkylguanine does not produce the same sensitization(Papirmeister et al, 1985). T71e sensitization was attributed by Papirmeister et al (1985)to N-3- alkyladenine DNA glycosylase. Both N-3-alkyladenine and N-7-alkylguaninedepurinate spontaneously (Papirmeister et al, 1985). These two alkylations may increasethe rate of spontaneous apurination by a factor of 105 to 106 (Verly, 1980). Thesubsequent action of an apurinic endonuclease may give rise to a DNA strand break(Papirmeister et al, 1985). In E.coll, 3-methyl adenine has been identified as a majorlethal lesion (Verly, 1980).

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Alkylation of the guanine 0-6 would be expected to alter the normal H-bondformation in DNA (Fig. 8). The formation of this product is known to cause miscodingresulting in GC to AT transitions (Gerchman and Ludlum, 1973; Lawley and Martin,1975). The miscoding occurs because the complementary base of 0-6 alkylguanine canbe uracil or thymine instead of cytosine (Gerchman and Ludlum, 1973). This lesion isknown to be a promutagenic lesion and procarcinogenic lesion and is also thought to be acytotoxic lesion (Kalamegham et al, 1988; Ludlum, 1987; Newbold et al, 1980). HeLAcell mutants deficient in the ability to repair 0-6-methylguanine DNA show an increasedfrequency of DNA strand breaks and these breaks are thought to be the cause of celldeath (Kalamegham et al, 1988). Ludlum (1987) and Tan et al (1987) have proposed thatthe unrepaired 0-6 alky!ation may give rise to crosslinks. A possible mechanism for theformation of crosslinks by - alkylguanine produced by CEES is shown in Fig. 9 (Ludlum,1987).

In summary, the DNA interstrand crosslink is thought to be the lesion mostlikely to be responsible for cytotozicity (Wilkinson, 1987). However, the role of theminor adducts is still unclear and may be of great importance (Ladlum, 1987). There isalso evidence that factors other than the interaction of the alkylating agents with DNAmodulate the response of the cell.

5.1.3 DfA Repair

Because DNA is alkylated by the mustards it is reasonable to expect that the cells DNArepair processes would influence the outcome. The moot important DNA repairpu,'hways depend on the excision of the alkylated base. The two main activities involvedare provided by DNA glycosylases which cleave the base-sugar bond of the alterednucleotide, and nucleases, which incise DNA by the specific cleavage of a phosphodiesterbond adjacent to a damased residue (Lindahl, 1982). The role of DNA repair is notcompletely clear, although a number of components have been examined. Fulldiacussions uf the mechanism of DNA repair may be found in Lindahl (1982), Roberts(1978), Strauss (1988), Tonini (1988) and Walker (1986).

Excision repair of bulky damage by UvrABC nuclease is thought to resultfrom detection by the enzyme system of distortions in the DNA helix (Watson et al,1987). Frankfurt (1987) has used antibodies to detect local DNA denaturation as a resultof alkylation by nitrogen mustard.

5.1.3.1 Ctsink Repair

The removal of DNA interstrand and DNA protein crosslinks has been observed by anumber of workers (Clarkson and Mitchell, 1981; Dean et al, 1986; Hansson et 91. 1987;Murray and Meyn, 1966). However, the exact mechanism by which the DNA interstrandcrosslinks are removed is unclear. The following mechanism has been proposed (Reidand Walker, 1969: Roberts, 1979):

a) two incisions are made on one strand near a cross-link;

b) the gap is widened by a nuclease, thus exposing a single-stranded region of DNA;

c) strand exchanges between homologous duplexes insert an intact base sequencecomplementary to the strand still carrying the partially excised cross-linkingresidue;

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d) when the twin helical DNA structure is restored, the remaining arm of the crosslink is excised.

As noted before (Sectian 5.1.2) Cells sensitive to the mustards ame notnecessarily deficient in cross-link repair. However, cells deficient in DNA cross-linkremoval may be sensitive to the miustards.

Ludlum (1987) has proposed that a 7-alhyl guanine DNA glycooylape capableof recognizing the 7-alkyLated guanine should be capable of repairing the crosslinks.

M.".3 Tbpoieoet U

Mwe enzyme topolsomerase 11 is involved in controlling the topological state of DNA(Wang, 1985). This enzyme is thought to alter the structure of the chromatin to allowaccess of mustard-induced monoadducta to repair enzyme* (Tan et al. 19M7)

Resistance to nitrogen mustard is associated with increased activity of DNAtopoisomerase 17, concomitant with decreased DNA cross-link formation (Tan at al,1967;1988B). 'Me increased activity is associated with increased transcription of thetopoisomerase 11 gene. Further evidence for the involvement of this enzyme is providedby the potentiation of the lethal effect of nitrogen mustard by novoblocint - atopolsomerase 11 inhibitor (Tan et al, 1988a).

Ll."3 04 alkylatiam rqusir

Bacte:,ai and mammalian cells are able to repair the 0-4 methylguanine adduct(Day at al, 1980; Dean et al. 1986a; Erickson et al, 1980; Kalamegharn et al, 1988;Karrah at al, 1979; Robins and Cairns, 1979). The enzyme responsible for the repair,04-methylguanine-DNA metlhyltransferase, is specific for methyl groups only at the 0-6position of guanine (Lindahl, 1982).

'Me alkyltransferase removes ethyl groups at a much slower rate than methylgroups (Lindahl, 1982) and is totally uinactive towards CEES alkylated DNA (Ludlum,1987; Ludlum et al, 1986). Ludlum (1987) and Tan et al (1987) have proposed that theunrepaired 046 alkylatlon may give rise to DNA interstrand cross-links (Section 5.1.2).

5.1.3.4 Poly(ADP-rlbOspolymerase

The enzyme poly(ADP-ribose)polymeraae (polyADPR-P) is indlapensible for DNA excisionrepair (Creissen and Shall, 1982; Durkncz et al, 1980; Hayaishi aad Ueda, 1982; Uedaand Haysishi, 1985). DNA damage wtimulates poly(ADP) ribosylatlon and a concomitantdecrease in NAD* MDurkacz et al, 1980; Ueda and Hayslshi, 1985). Inhibition ofpolyADPR-P inhibits the ligation step (DNA ligase 11) in DNA repair (Creissen and Shall,1982). The NAD+ depletion and cell death induced by the DNA alkylating agentdimethylsulphate are prevented by polyADPR-P inhibitor@ (Stubberfield and Cohen,1988).

Dean and Fox (1984) and Papirmeister et al (1985) huve proposed ageneralized disturbance of cell metabolism involving nucleotide pool imbalance as a

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consequence of intoxication by mustards. According to Papirmeister et al (1985) thesequence of events following exposure to mustard is as follows:

1. DNA aikylation;

2. spontaneous and enzymatic depurination resulting in apurinic sites and DNA breaks;

3. activation of poly(ADP-ribose)polymerase by the accumulation of DNA breaks;

4. lowering of cellular NAD+;

5. inhibition of glycolysis and accumulation of GOP;

6. stimulation of NADP-dependent pentose phosphate cycle;

7. protease release and

S. pathological changes.

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 humanskin Fafts and leukocytes resulted in a dose-dependent decrease of up to 40% in theNAD levels (Meier et al, 1984; 1987; Papirmeister et al, 1985). More importantly, inthe leukocyte work, the decrease in NAD÷ only occurred in those cells (lymphocytes)containing poly(ADP-ribose)polymerase (Meier et al, 1987). Finally, inhibitors ofpoly(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 beestablished. Although some monofunctional alkylating agents are known to stimulatethe synthesis of poly(ADP-ribose) (Juarez-Salinas et al, 1979), Graham and Fox (1983) didnot observe such an increase in nitrogen mustard treated cells. Secondly, although theNAD+ 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 ofpoly(ADP-ribose)polymerase is also not ideal since these compounds also inhibit the DNArepair required for the cells to recover (Meier et al, 1984). Also, as pointed out byWhitfield (1986) other inhibitors of NAD. synthesis such as 3-acetylpyridine and6-aminopyridine should also be vesicants.

5.1.3.5 Deparination

As indicated in Section 5.1.2 alkylated bases may undergo spontaneous depurination.Depurination may also be enzymatically mediated. Several enzymes, DNA glycosylases,exist which are capable of breaking the sugar-base bonds in DNA and whhich have doublestranded DNA, as the preferred substrate (Lindahl, 1982). Distinct er.zymes 'exist .or theremoval of 3-methyladenine and 7-methyl guanine (Lindahl, 1982). Ludlum (.1987) hasproposed that a 7-alkyl guanine DNA glycosylase exists which is carable of recognizing7-ethylthioethyl deoxyguanine in CEFS treated DNA and releasing it. The apurinic sitesgenerated by the action of these enzymes should be rapidly repaired by an excision-repairprocess (Lindahl, 1982).

17

L.2 Ghatathim

Glutathione (L1y-glutomyl-L-cysteinyl-L-glycine) occurs in high concentration in cellsand acts as a redox agent (Scott and Eagleson, 1988). The Orrenius theory of toxicitysuggests that depletion of glutathione (GSH) is one mechanism by which compounds killcells (see Whitfield, 1987). The involvement of GSH in the toxicity of nitrogen andsulphur mustard is still unclear. Several groups have reported increased levels ofsulfhydryl groups in cells resistant to phenylalanine mustard (Begleiter at al, 1983;Suzukake et al, 1982). In addition, the formation of a diglutathione conjugate betweenphenylalanine mustard and GSH has been demonstrated (Dulik et al, 1986). Reduction ofGSH levels by buthione sulphoximine or dietary manipulation sensitizes cells which are •resistant to phenylalanine mustard (Somfal-Relle et al, L984; Suzukake et al, 1982) whileelevation of GSH by diethylmaleate increases resistance (Taylor and Brown, 1988).

The situation for nitrogen mustard is unclear. Robson at al (1987) founddecreased DNA interstrand cross-link formation accompanied by elevated GSH andelevated glutathione S-transferase activity in Chinese hamster ovary cells resistant tonitrogen mustard. Conversely, Hansson at al (1988) observed increased nitrogen mustardtoxicity accompanied by increased DNA interstrand cross-linking in human melanomacells 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 insensitive cells, the reduction of GSH by buthione sulphoximine did not affect thecytotozicity (Tan et al, 1987). In addition, Walker 258 cells resistant to nitrogenmustard exhibited reduced glutathione reductase activity (Dean at al, 1986).

5. Memoi'ne Effects

Nitrogen mus-Ard is known to have a number of effects on cellular functions associatedw'.h the cell membrane. Treatment of tumour cells or erythrocytes results in decreasedrotational diff ision of membrane components which is ascribed to the formation ofprotein cross-links (Doppler at al, 1985; Grunicke at al, 1985; Levy 1965).

The use of 86Rb+ as a K+ congener has shown that nitrogen mustarddecreases the furosemide sensitive K+ transport, which is mediated by a Na+/K+/CI-cotransporter (Baxter et al, 1982; Doppler et al, 1985; Grunicke at al, 1985; Wilcockand Hickman, 1988; Wilcock at al, 1988). It is important to note that themonofunctional. mustard (2 chloroethyl)dimethylamine has no such effect (Baxter, 1982ýWilcock at al, 1988). The ouabain sensitive Na pump is only affected at relatively highconcentrations of nitrogen mustard (Baxter at al, 1982; Doppler et al, 1985).

The significance of these observations is unclear. The fact that furosemidedoes not affect the cell proliferation indicates that the inhibition of K+ transport bynitrcgen mustard is not sufficient to explain its effects (Doppler et al, 1985; Wilcockat al, 1988). It ailso remains to be established whether the effect of nitrogen mustard isdirect or indirect (Wilcock at al, 1988).

The trifunctional mustard tris(2-chloroethy!)amine (HN3) has been found toalkylate spectrin and cross-link it to itself and other membrane proteins in humanerythrocytes (Wildenauer and Wegar, 1979). The result of the alkylation is fixation ofthe cell shape and protection against metabolically induced chances of shape.(Wildenauer at al, 1980). Spectrin forms the basis of the erythrocyte membrann skeleton(Bennett, 1985). No reaction between HN3 and phospholipids was detected in thisexperimental system (Wildenauer et al, 1980).

18

L.4 C•ptoplasmic Effects

In addition to the effects on nucleic acids and the cell membrane, the effects oncytoplasmic components have also been studied.

Wildenauer and Weger (1979) and Fung et al (1975) have observed thealkylation of haemoglobin by nitrogen mustard. In human erythrocytes the formation ofdimers was observed (Wildenauer and Weger, 1979). In isolated sickle cell haemoglobinalkylation at the 02 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 oflysosonal enzymes in vitro but only at extremely high concentrations (Gross et al,1981). In vivo, treatment of rats with nitrogen mustard also indicated that theseenzymes are unlikely to be involved early in the response of the cell (Cohen, 1978).

Walker ascites -carcinoma cells resistant to phenylalanine mustard exhibit adecrease in the activity of a low Km adenosine 3,5i-monophosphate phosphodiesterase(Tisdale and Phillips, 1975a). 'In sensitive cells, this enzyme is inhibited by treatmentwith chlorambucil and the concomitant increase in the level of cAMP is correlated withinhibition of cell growth (Tisdale, and Phillips, 1975b).

6. PROTECTION

The pretreatment and therapy available for mustard poisoning have been reviewedrecently by Whitfield (1987) and the results of that review will be presented here. Thecompounds considered by Whitfield are shown in Table 1 and a summary of thecharacteristics of potentially useful compounds in Tables, 2 and 3 (from Whitfield, 1987).

Sodium thiosulphate nppears to be the most effective pretreatment currentlyavailable (Fasth and %)rbo, 1973; McKinley et al, 1982; Whitfield, 1987). However,posttreatment appears to be ineffective (McKinley et al. 1982). Sodium thiosulphate isalso effective in reducing the skin damage produced by nitrogen mustard in mice(Dorr et al, 1988). However a 200:1 molar excess of sodium thiosulphate was requiredand delaying the treatment dramatically reduced its effectiveness (Dorr et al, 1988).The use of thiosulphate combined with other drugs such as ,examethasone, cortisone,promethazine, heparin and vitamin E is effective in limiting , th. skin damage andincreasing the survival of experimental animals exposed to silphur mustard (Vogt et al,1984; Vojvodic et al, 1985).

7. DISCUSSION.

It is appropriate in discussing the mechanism of action of ritrogen and sulphur mustard tobear in mind the opinion of Farber (1971):

19

/

"It is eminently clear that virtually every pathologic process and dinse..must have a clearly defined biochemical bane before we can admit to mysignificant understandi.g of that entity. Yet it is also clear that mostnaturally-occurring disea3e, be it induced by chemicals, viruses, bacteria,dietary deficiencies or other environmental alterations, is extremelycomplex at the level of the chemistry of the cell and may often beimpossible to unravel with current knowledge. This is in part due to theexistence of many interlocking regulatory control mechanisms which allow'for integration and modulation of the metabolic activity of the cell.

Certainly, in the case of ,the mustards, the exact biochemical bass han yet tobe established without doubt. The observation that proliferating cells are spmeciallysensitive, that the mustards are mutagenic and carcinogenic and that the earliest visibleeffects ,ccur in the nucleus implicate DNA in the mechanism of action of thesecompounds. It is now well established that sulphur and nitrogen mustard elkylate DNAand produce interstrand, intrastrand and DNA-protein cross-links as well asmonofunctional adducts. However, the contribution of these modifications to lethalityis unclear. Opinion in the literature ranges from positive assertions that the DNAinterstrand cross-link is the lethal lesion, to assigning lethality to general disturbance ofDNA metabolism. Also, as Hanason et al (1987)'have suggested, the position of thecross-link in the genome may be more important than the absolute number of cross-links. The monofunctional adducts are also attracting greater interest and some ofthese remain to be identified. The contributions to lethality of the RNA alkylations andthe RNA-protein and DNA-protein cross-links'have yet to be established. Thetranscription of DNA alkylated by nitrogen mustard produces RNA of reduced chainlength (Kann and Kohn, 1972). The physiological consequences of this disturbance ofRNA metabolisim have yet to be established.

In the past, alkylation of protein a a significant contribution to the lethaleffects of the mustards has been discounted on theoretical grounds (Roberts, 1978; Ross,1962). Roberts (see Farber, 1971) has stated that it the level of alkyintion with sulphurmustard which inhibits DNA rynthesis and cell division, the level of protein alkylation issuch that only one protein molecule in 2000 (assuming a molecular weight of 100,000) willbe alkylated. However, alkylation of a critical protein may well be important. Forexample, Wilcock at al (1988) have demonstrated on effect of nitrogen mustard on the:Na+/K+/Cl- cotransporter, although it remains to be demonstrated whether or not this,effect is secondary to the effect on DNA.

The observation that a number of DNA repair enzymes modify the lethaleffects of the mustards is compelling evidence for a central role of DNA in themechanism of action of these compounds. However, several studies have hmwn nodifference between sensitive and resistant cell strains in their ability to repair DNAinterstrand cross-links. This does not indicate that DNA damage is not the lethal lesionIn these particular cells, but does indicate that other factors may be irivolved. Theobservation that cells already deficient in DNA repair are more sensitive to alkylatingagents (Meyn and Murray, 1984) reinforces the central role of DNA.

Further evidence for the involvement of DNA in the lethality of the mustardsis provided by the observation that caffeine enhances the lethality of nitrogen mustard(Murnane et al, 1980; Roberts and Kotsaki-Kovatsi, 1986). This enhancement incorrelated with the production of double-strand breaks in the DNA (Roberts and Kotmaki-Kovatsi, 1986) and fragmentation of the nucleus (Lau and Pardee, 1982). Caffeine is

20

thought to reverse the inhibition of replicon initiation and thus allow the synthesis of newDNA on a damaged template (Carr and Fox, 1982). The delay in G2 is the response ofthe cell to the detection ef the DNA damage. Tobey (1975) has proposed that asurveillance mechanism operates in G2 to ensure that damaged DNA is repaired prior tomitosis. The results of Lau and Pardee (1982) are particularly persuasive of the centralrole of DNA damage because the effect of caffeine was demonstrated using doses ofnitrogen mustard which allowed 85% of the cells to survive in the absence of caffeine.The involvements of topoisomerase II also indicates a role for DNA in lethality.

It is also clear, as discussed above, that otl'er factors are involved in thelethal effects of them compounds. The evidence in favour of the involvement of GSH isstrong. Inhibitors of the choline, transport system, which transports nitrogen mustard

into the zell, also reduce the level of DNA croso -.inking and protect the cells (Doppler etal, 1988). Conversley, cells with a defective choline carrier display reduced DNA cross-links and reduced sensitivity to nitrogen mustard.

The question also arises whether the mustards indiscriminately alkylate allavailable sites rather than preferentially alkylating nucleic acids, The study of Ritter etal (1988) is important in addressing this question. These workers found that nitrogenmustard could be recovered from Ehrlich cells, an observation inconsistent with theconcept of instantaneous covalent binding of nitrogen mustard. Their uptake datasuggest a two stage process whereby the nitrogen mustard is bound to a carrier andmaintained is such a state that 2/3 of the unbound mustard is tramsferred to nondiffusiblemolecules or nonexchanging compartments and 1/3 is available for return to the externalmedium. Furthermore, vincristine decreases the effects 6f nitrogen mustard, suggestingthat the intracellular transport of the dv.,g is an important' factor in its mechanism ofaction (Ritter et al, 1988). Finally, Ca increases the Iftake of nitrogen mustard butdecreases its toxicity, prompting the suggestion that Ca acts on an intracellularmechanism which directs the nitrogen mustard away from the nucleus (Ritter et al,1988).

The fact that nitrogen mustard can exist intact within the cell suggests thatit can exist in a sequestered form (Ritter et al, 1988). Rutman et al (1969) also observedthe retention of functional alkylating activity bound tc DNA. Their assumption thateither an unreacted chloroethyl group or aziridinium ion was sequestered within the DNAstructure provides some evidence for the ability of nitrogen mustard to exist in asequestered form.

In summary, the available evidence indicateq that DNA is the major target ofsulphur and nitrogen mustard but other factors modulate the final effect. The exactcause of cell death is unknown. Hypotheses vary from the release of proteases(Papirmeister et al, 1985) to the ill-defined concept of 'unbalanced growth' or 'out ofphase synthesis'. The consequences of many of the effects, for example RNA alkylationand the function of DNA-protein and RNA-protein cross-links, remain to be elucidated.

21

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33

TABLE 1

Poraible Pretretment and Tberspy CoinpoumchConsidered by Whitfield (1987)

Dithiocarba mates

Cysteine and Cystine

Glutathione

Radioprotective Agents

Anti-arsenicals and other 17hiols

7hioeulphqte and its Precursors

7tiophosphonates

TABLE 2

Summary of Characteristics of Compounds Potentially useful asPretreatment@ against H and HN2 Poisoning

Property S2032- DMDTC DEDTC Disulfiram Cysteine GSH WR2721

Maximum dose 3.0 5 1.0 >10 mg 1.2 4.0 >0.6(mg/kg)

Side Some Possible delayed neuro- Nausea at very ToxicEffects nausea pathy and others high doses

Period of 10 Mins 1 1 24 0.5 un- >0.25effective- knownness (hrs)

Route of ip in ip ip Oral, ip, iv Oral, ipadministra- large 5 ml/kg 5 ml/kg possibly iption vol implant

Enters cells No Yes Yes, Yes Yes Slowly Yes

Competition 27,0(0 40,300 33,000 Unknown 1.050 <1,050 Unknownfactor

Proven Yes Promising No Yes No Yeseffective (N and (H) (HN2) (H) (HN2)

HN2)

Available Yes Yes Yas Yes Yes Yes Yescorn mercially

Reproduced from Whitfield (1987)

TABLE 3

Protection indices of various sulphur-containing compoundstowards murine stem cells treated in vivo with HN21. Maximum tolerated dose. An MT'D of 10 mg/mouse

corresponds to a dose of 400-500 mg/kg.S(Thiol + 1tN2)

2. Protection index - S(hn2 alone)

S - number of surviving femur stem cells.

Compound MATD 1 Protection

(mg/mouse) index 2

WR-3689 15 159

WR-2721 15 44Aminoethylcysteine 80 27N-acetylcysteine 8 26

Glutathione 60 22Cysteine 8 15

2-a minoethylisothiourea (AET) 10 14Sodium thiosulphate 50 12.2-aminoethanol 3 6.6

Cysteamine 8 3.5

WR-638 20 3.4

Disulfiram 6 1.1Ethylamine 1 1.0

2-mercaptoethanol 3 0.6Dimethylsulfoxide 250 0.49

Reproduced from Whitfield (1987)

CH2 CH2 C1/S

CH2 CH2 Cl

CH2CH2 0H/S 2

CH2 CH2 Cl

Figure 1 Sulphur mustard (1) and half mustard (2).

Cf2 CH2Cl

H3C-N

CH2CII2CI

CH2 CHCHCC

O NH 2 CH2CH2CI

HOOCCH2 CH2CH2 N,, ~ C 2 HC

Figure 2 Nitrogen mustard and derivatives.1. Nitrogen mustard (11N2)2. Nornitrogen mus.ard3. Phenylalnnine mustard4. Ghlutambucil

CH 2CH 2OH

/CH2CH2 -S

/ ICH2 CH.,OH

SCHCH 2CH21

Figure 3 Higher homologues of sulphur mustard.

CH2CH3

S.1

CH2CH2C1

/CH3

NH2CON 2.

NO

/ NOHN= C 3

CH3SO2OCH3 4

Figure 4 Additional alkyla ting agents:1. Chlorvethyl ethyl sulphide2. N-methvl-N-ni trosouren3. N-methyl- N nitro-N-nirrosog'unnidine4. methyl methane suiphonate.

Fkgure 5 Phases in the life cycle of a cell. M1 is the mitotic phase and S the DNAsynthetic phase. The two gap phases are Gi (pre-DNA synthesis) and G2(period between DNA synthesis and mitosis). Th)e relative lengths of thevarious phases differ between cell types.

The major purines The major pyrimidines

AdenineCytosine

NHH2c NH

I I CH1 .2 4C"\'

C" N IC N 4C HN N ecH

H

Guanine Uracil

0I 0

"'NHNI Nc \1 11 CH HiN(4NCH

HN-C 4 -ecý / IN N OmICz OC

H.

Thym me

0

HNI('-C-CH,

NH

Flgure 6 11e major purines and pyrimidine,.

CH2CH2SCH2CH3

0

EN N

HN. N NH H

0

HN -N -CH2CH 2NHCH2CH 2C1

HN N NH H

0 0.

HN NCHzCH 2NHCH2CHZN HN

HN <N N'K N N HNH H H H

Figure 7 Products from the alkylation of DNA bynitrogen njustardl and CEES:Upper - OPethylthioethYl guhnineCentre - 7 ethyl thloethyl guanine'Lower - N, N-bis (247-~guaninyl)-ethylb inehll.

NH2 N• ,, 2 ............ 0N

N ............... HN N

0 HN H

CH3

NH2 0 N

N ....... HN

H N< HN> H0 ................. HH

Figure 8 Altered 1l-bonding by formation of 06 alkylguanine. Guanine normally pairswith cytosine (top). Methylatiun disrupts the normal H-bonding (bottom) andallows pairing with thymine.

0

N 0 0

H i

* o

00Ni 00

Ludlum, 1987).

/

/

sscUI1Y CLASSIFICATION OF n31s nO3 UNCLANED

DOCUNNT? CONTROL DATA 3SN3T

3ZORT 30. AN 00. 3SU? s-CU1?T CLASSIVICATION

MRL,-TR-89-24 AR-006-717 Unclassified

A literature review on the mechanism of actionof sulphur and nitrogen mustard

&tU58GeO) IUORATS AUTNOR

W IGtaterials Research LaboratoryP.J. Gray PO Box 50

Ascot Vale Victoria 3032

RE1PORT DAS 13 5. TANS N0O

July 1989 ARM 881139 ARMY

lILS NO. aLPm.AM

G6/4/8-3742 149 46

CLASSInCA*•IXN/L!NITATION a2, Z3 DATI CLASIFICAI.O/R8L/• Sa AUTWORITYChief, Protective ChemistryDivision, MRL

llNART DZITRIBUTIOt

-Approved for public release

Announcement of this report is unlimited

Mustard Agents Nitrogen Mustards Chemical AgentsVesicants

suanZC GmRWS 0074D

The literature describing the mechinism of action of sulphur and nitrogenmustard is reviewed. The conclusion drawn is that the available evidence suggests thatDNA is the most important molecular target of these compounds. However, the finaloutcome of poisoning is modulated by other factors such as DNA repair, glutathione levelsand drug transport.

8UCU'ITT CLASSIFICATION OF TNIS PA0s

UNCLASSWWD