The genetic toxicology of nitrogen and sulphur mustard

131

Mutation Research, 75 ( 1 9 8 0 ) 1 3 1 - - 1 6 8 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

THE GENETIC TOXICOLOGY OF NITROGEN AND SULPHUR MUSTARD

M A R G A R E T F O X a n d D. S C O T T

Paterson Laboratories, Christie Hospital and Holt Radium Institute, Withington, Manchester M20 9BX (United Kingdom) ( R e c e i v e d 19 M a r c h 1 9 7 9 ) (Revision received 10 July 1 9 7 9 ) ( A c c e p t e d 12 J u l y 1 9 7 9 )

Contents 1. 2. 3. 4. 5. 6. 7.

Introduction .............................................. Usage in m e d i c i n e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . O t h e r uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C h e m i s t r y a n d overall levels o f a l k y l a t i o n in D N A . . . . . . . . . . . . . . . . . . . . . . Repair of damaged DNA and effects on DNA synthesis . . . . . . . . . . . . . . . . . . Cross-sensitivity a n d resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mutagenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. M i c r o o r g a n i s m s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b. Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c. D r o s o p h i l a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d. M a m m a l i a n cells in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . e. G e r m cells o f m a m m a l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i. A n t i s p e r m a t o g e n i c e f f e c t s and d o m i n a n t lethals . . . . . . . . . . . . . . . . . . ii. T r a n s l o c a t i o n s a n d recessive visibles . . . . . . . . . . . . . . . ........... 8. C y t o g e n e t i c a l l y d e t e c t e d e f f e c t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a . Clastogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i. Types of aberration ..................................... ii. Lesions and m e c h a n i s m s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii. D o s e - - r e s p o n s e r e l a t i o n s h i p s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv. H u m a n cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b. S i s t e r - c h r o m a t i d e x c h a n g e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c. C h r o m o s o m e stickiness, segregational errors a n d n u m e r i c a l a b n o r m a l i t i e s . . . .

132 133 133 134 136 138 140 140 144 145 146 148 148 150 151 151 151 151 153 153 153 153

AML, acute myeloid leukaemia; asn, asparagine; CEES, 2-chloroethyl ethyl sulphide; CFA, colony-foisting ability; chl, chlorate; EMS° ethyl methanesulphonate; gal, galaetose; HIPA, (D(+)-cl-hydrazino-imidazolepropionic acid; HN2, nitrogen mustard; IUdR° 5-1odo-2-deoxyuridine; IUdR r, 5-iodo-2-deoxyuridine resistant cells; lac0 lactose; LEC, lowest effect concentration; MDMS, methylene dimethanesulphonate; MMS, methyl methanesulphonate; MNNG, n-methyl-n-nitro-n-nitrosoguanidine; MOPP, nitrogen mustard, vincristine° prednisolone0 proearbazinc; REC, tad-equivalent" chemical; SM, sulphttr mustard; TdR, thymidine; TdR r thymidineresistant cells; TEB° triePoxybutane; thr, threonine; TK, thymidLne klnase; 244,6 triCl PDMT, 1-(2,4°6-trichlorophenyl)-3-3-dimethyltrlazine; UDS, unscheduled DNA synthesis; XP, xeroderma pigrnentosum; YR and YS, Yoshlda sarcoma cells which are resistant (R) or sensitive (S) to nitrogen and sulphur mustard. Abbreviations:

132 9. Acute toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10. Carcinogenicity ............................................ 11. T e r a t o g e n i c i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12. Germ-cell hazard: problems of extrapolation to man 13. Conclusions ............................................... Acknowledgements ............................................. References ..................................................

....................

155 155 156 157 159 160 160

1. Introduction In contrast to many of the chemicals recently considered in "Reviews in Genetic Toxicology" there can be no doubt as to the mutagenicity of nitrogen and sulphur mustard and their derivatives. Chemicals of this type were amongst the first shown to possess genetic activity [6,8,9]; between 1946 and 1950, mustard gas {sulphur mustard, SM) and nitrogen mustard (HN2) were shown to be capable of inducing mutations in Drosophila [6,8,9] and in Zea mays [83]. The demonstration of their mutagenic effects was paralleled by direct observations of cytological damage in a variety of plant [56,119] and animal material [17,18]. Subsequently, both compounds were shown to be mutagenic in other species, including bacteria, fungi and rodents. Such studies up to 1962 have been reviewed by Wheeler [228]. These early demonstrations of the mutagenic activity of mustards strongly implicated reaction with nucleic acids. However, the possibility that their cytotoxic effects were due to unspecified metabolic disturbances was being considered by biochemists throughout this period, and extensive studies of the effects of these agents on many aspects of cellular activity were reported [228]. Mustards were shown to affect the levels and activity of a wide variety of enzymes in many tissues in a wide range of organisms and, in spite of conflicting results, Wheeler [228] concluded that there was some correlation between the effects of these compounds on glycolytic enzymes and their carcinogenic, vesicant and carcinostatic properties. A feature common to early reports of the biochemical effects of these drugs on protein synthesis and enzyme activity was that doses many times higher than those necessary for inhibition of mitosis were used. Thus, whilst it was realised that mitotic activity was highly sensitive to these agents, effects on this process were not generally considered by many workers to be the result of direct interaction with nucleic acids. Thus, the relationship between inhibition of cell division, inhibition of DNA synthesis, the ability of these agents to cross-link DNA and their mutagenic effects were not fully appreciated up to 1962, although the involvement of DNA in mutagenesis had been suggested in 1960 [137]. More direct evidence that the cytotoxic effects of nitrogen mustard were due to alkylation of nucleic acids was obtained by studies on transforming DNA. Inactivation of transforming DNA of Pneumococcus and Haemophilus influenzae [235--237] by nitrogen and sulphur mustards was demonstrated after exposure to doses which were within the range of those which produced measurable nucleocytotoxic effects on animal tissues in vivo. These experiments, which strongly suggested that nucleic acids were a major site of attack

133

of compounds of the mustard type, led to a series of studies of the chemical interactions of a variety of labelled compounds with DNA and RNA in vitro, and marked the beginning of our present-day understanding of their cytotoxic and mutagenic activity at a molecular level. Several factors influenced us in the choice of material for the present review since to consider the biological effects of the many thousands of nitrogen and sulphur mustards that have been synthesised [31,32] would be a formidable task. Firstly, there is already a vast literature concerned with the antitumour effects, molecular biology, chemistry and pharmacology of many of these derivatives in both experimental animals and man [see refs. 51,61,62,127,138, 163,177,183,203,228]. Secondly, much of the data on the cytotoxic effects of these derivatives has confirmed an early view expressed by Bodenstein and Goldin [17,18] that many of the responses provoked by the various kinds of derivatives can be brought about by an appropriate concentration of any one particular compound [120,149,150]. Thirdly, the genetic and allied effects of a large number of alkylating agents including nitrogen and sulphur mustard have been extensively discussed [7,127,138] and the genetic toxicology of some of the clinically more widely used nitrogen mustard derivatives, cyclophosphamide, isophosphamide and trophosphamide has also been the subject of a recent review [146]. With these factors in mind we have restricted ourselves mainly to consideraton of the genetic toxicology of the two parent compounds. We have included considerable discussion of data on cytotoxicity since many of the phenomena associated with cytotoxic effects, e.g. growth inhibition and inhibition of DNA synthesis have been proposed as potentially useful short-term tests for screening mutagenic chemicals in mammalian cells [29]. In discussion of both the cytotoxic and mutagenic effects we have attempted to interpret the data in the light of our present-day knowledge of the reactions of both sulphur and nitrogen mustard with DNA and the influence of repair processes on the biological consequences of such reactions. 2. Usage in m e d i c i n e Sulphur mustard was first used clinically in the treatment of solid tumours in 1931 [1]. However, the toxic and vesicant effects of this compound precluded any further clinical usage. The first reported clinical use of nitrogen mustard (HN2) was in the treatment of Hodgkin's disease and certain other lymphomas by Gilman and Phillips in 1946 [85]. Since then, the hydrochloride of nitrogen mustard has been widely used in the treatment of this and other types of neoplastic disease in man. Clinical trials have also been conducted to assess its usefulness in the treatment of rheumatoid arthritis and as an immunosuppressant in other non-malignant conditions [142,187,209]. The hydrochloride salt has also been used in veterinary medicine for the treatment of malignant disease [216]. 3. Other uses

Nitrogen mustard has had limited use as a cross-linking agent in textiles [176,184], as a sizing agent for cotton y a m [125], as a fungicide [164], and as

134

an antiviral aerosol disinfectant [211]. The hydrochloride salt has also been tested as a cross-linking agent in manufacture of ion~exchange fibres [186]. Mustard gas (sulphur mustard) was of course used as a vesicant in chemical warfare during World War I [225]. Nitrogen mustard was also produced as a possible vesicant but never used [102]. 4. Chemistry and overall levels of alkylation in DNA The chemistry of nitrogen and sulphur mustard reactions has been studied in some detail [127,183]. Briefly, HN2 (di-2-chloroethyl methylamine) in aqueous solution cyclises by a fast SN,-type mechanism to an immunium form / CH2CH2C1

SN~ ~a,t

CH3N

\CH2CH2CI

/ CH2CH2C1 CH3N + x/ \ CH2--CH2

ISN /CH2CH2CI

CH3N\cH2CH2 X which will react more slowly with a nucleophilic site (X-) by an SN2 reaction. Sulphur mustard, on the other hand, only forms the analogous sulphonium ion in small amounts, the bulk ionising to the carbonium ion by a fast reaction. The carbonium ion then reacts with a nucleophilic site by a fast SNI reaction, the half-life of the whole reaction in water being approximately 3 min at 37°C. S/CH2CH2C1 tas~ C1CH2CH2SCH2CH2 + X- ~ C1CH2CH2SCH2CH2X \ CH2CH~C1 Thus the rate of reaction of sulphur mustard will be essentially independent of the concentration of nucleophiles present unlike the second stage of the nitrogen mustard reaction. Some of the chemical and physical properties of sulphur mustard, nitrogen mustard and its hydrochloride are summarised in Table 1. Reactivity of the guanine moiety in nucleoproteins towards mustard gas is now well established; definitive evidence has been obtained that the N-7 position is the major site of attack, and 7~dkyl guanine identified as the major product [127]. The two reactive chloroethyl groups of mustards allow the formation of cross-links between guanines on the same or on opposite DNA strands and this reaction product has been isolated and identified as diguanin-7ylethylmethylamine [127]. Such cross-links appear to constitute approx. 25% of the total alkylations, the rest being in the form of monofunctional reaction products. The relationship between dose and level of alkylation is linear [35], and the percentage of cross-links relative to total alkylations is independent of dose [13,127,128,130]. The proportion of inter- to intrastrand cross-links has been the subject of some debate. Lawley [127,130] however, concluded that approximately 1/3 of cross-links i.e. 7--10% of total alkylations were interstrand. 7-Alkyl guanine has been shown to be the major site of DNA reaction

135 TABLE 1 CIIEMICAL AND PHYSICAL PROPERTIES a OF SULPHUR MUSTARD, NITROGEN MUSTARD AND ITS HYDROCHLORIDE

Sulphur m u s t a r d

Nitrogen mustard

Chemical abstracts number

505-60-2

Mol. w t .

159.1

156.1

192.5

Description

Colourless, oily liquid

Liquid, faint fishy odour

W h i t e hygroscopic crystals

--60°C

109--111Oc

Melting point

13--14°C

51-75-2

Nitrogen mustard (hydrochloride)

Boiling p o i n t

2 1 5 - - 2 1 7 ° C at 7 6 0 m m H g

Density

d 20 1.274 (liquid)

d 2S 1 . 1 1 8

Solubility

S p a r i n g l y in w a t e r ( 0 . 6 8 g/1 at 2 5 ° C ) ; s o l u b l e i n fat, fat solvents and other common organic solvents

S l i g h t l y soluble in water; miscible with many organic solvents a n d oils

55-86-7

87°C at 18 mm Hg

S o l u b l e i n w a t e r (1 g/ 1 0 0 m l ) a n d in e t h a n o l

a Data from ref. 216.

of a large number of other alkylating agents, with other bases, including the 3-position of adenine and the 06 position of guanine being alkylated to a lesser extent. Alkylation at the 1 and 7 positions of adenine and the 04 of thymine and of phosphotriesters has also been demonstrated and the subject has been extensively reviewed [177]. No detailed study of the relative frequency of alkylation of different DNA bases by mustards appears to be available. In general, agents with an SN2-type reaction mechanism, e.g. nitrogen mustard, appear to react more exclusively at the N-7 position of guanine and are less likely to attack O sites than agents reacting by SNl-~pe mechanisms. Thus the spectrum of DNA reaction products resulting from nitrogen and sulphur mustard alkylation may well be different [see ref. 177]. Values for overall extent of reaction and number of cross-links (inter- and intrastrand) in DNA after exposure to sulphur or nitrogen mustards are summarised in Table 2. These data indicate that repair-proficient bacterial and mammalian cells can withstand considerable amounts of alkylation of their genome at D37 doses. The values for moles alkylation/genome are obviously dependent on genome size, and when alkylation levels are calculated on the basis of moles alkyl/107 daltons a value of the order of 1 mol alkyl/30 000 moles DNAP at a D37 dose is obtained, indicating that in general the reactivity of mustard with DNA differs little between species. However, in repair~tefective strains, e.g.E, coli, T even phages and mustard~ensitive Yoshida sarcoma cells the levels of overall alkylation and of DNA cross-links at Ds7 doses are significantly lower with values ranging from 2 to 50 cross-links/genome. If onethird of these cross-links are interstrand then 1--3 of such cross-links, if unrepaired, must constitute a lethal event (see Discussion by Loveless [137, 138] of phage inactivation kinetics, more recent work [27,137,231] on phage, and the extensive discussion by Roberts of cytotoxic effects of cross-linking agents [177]).

136 TABLE 2 LEVELS OF DNA A L K Y L A T I O N A F T E R EXPOSURE TO SULPHUR OR N ITR O G EN MUSTARD Organism

D O dose a (/Jg/ml)

Moles a l k y l l genome

Cross-links/ genome

Genome size (moles DNA P)

References

10.5 10.5 30.0 8.0 0.8 2.0 6.0 0.15

12 40 8 320 43 126--180 378--540 2.3 X 104

3.6 X 105

34, 166 130 34, 34, 34, 34, 54,

64 9 24 72 5 700

0.02

4.0 × 104 2.4 × 104

10 000 4 000

2.3 × 1010 2.0 × 1010

174 54

0.1

4.2 × 103

1 200

2.0 × 1010

182

0.0025

2.0 × 102

50

1.0 × 1010

201

2.5

2.0 X 103

500

1.0 X 1010

201

Sulphur mustard b

T4 phage T 2 phage T 7 phage E. coil B/r E. coil Bs-- 1 E. co li B E. coli B/r

He La cells Mouse L cells L 5 1 7 8 Y mouse lymphoma V79 Chinese hamster YS (Yoshida sarcoma) YR (Yoshida sarcoma)

2--3 8

3.6 9.0 9.0 9.0 9.0 3.0

X X X X X X

104 106 106 105 106 1010

35

35, 128, 129 35, 128, 129 35, 1 2 8 , 1 2 9 35, 128, 129 180, 181, 182

Nitrogen mustard c

7.8

T 7 phage Mouse: LES ( t u r n o u t cells) L2RA (turnout cells) Hamster: plasmocytoma in vivo Ehrlich a s c i t e s

17

4.5

3.6 X 104

24, 223

0.1

2.5 × 104

2 500

2

X 1010

44

1.0

2.5 X 105

25 000

2

X 1010

44

--

1.6 × 104

4 000

1 . 0 X I 0 I0

188

--

3.3 X 104

8 I00

1.0 X 1010

229

a DO dose required to reduce survival 1/e on e x p o n e n t i a l part of the dose--response curve. b ExposUre times varied from 15 to 45 rain. 50% h y d r o l y s i s t i me of SM water is ~ 3 rain [183], t h e r e f o r e concentration

~" d o s e .

c Biological half-Hfe is 45 min in tissue culture m e d i u m with serum (our unpublished observations), a n d less t h a n 60 rain in vivo [ 1 8 3 ] .

5. Repair of damaged DNA and effects on DNA synthesis The effects of mustards on DNA synthesis and the relationship between initial levels of reaction, numbers of cross-links and cytotoxicity have been reviewed recently [177]. The degree of cross-linking has been measured by a variety of methods, e.g. use of labelled compounds [34,35,128,129,130,223, 188,229], the ability of mustard-treated DNA to reversibly renature [108,118, 222] and by analysis of treated DNA in CsC1 density gradients [13,223,230]. The degree of initial cross-linking has been related in phage [24,34,35,130,166, 223], bacteria [128,129] and mammalian cells [44,54,180--182,201,188,229] to effects on DNA synthesis and survival of colony-forming ability. Such studies have led to the general conclusion that the cytotoxic effects of

137

mustards are related to their ability to cross-link DNA and hence inhibit its replication. Neither RNA synthesis nor protein synthesis are inhibited at doses which inhibit DNA synthesis, and current evidence indicates that recovery of the rate of DNA synthesis is associated with the removal (probably enzyme mediated) of diguanyl cross-links and monofunctional alkylation products (see Roberts [177] for review of evidence). The loss of alkylation products is associated with non-semi~onservative DNA synthesis (repair replication) in both bacteria [28,92,94,194] and mammalian cells [180,182]. That both mono- and bifunctional adducts are lost from DNA of mammalian cells is evident, since equal amounts of induced repair replication were observed after exposure of HeLa cells to equitoxic doses of sulphur mustard and "halfsulphur mustard", its monofunctional analogue [179]. There is however some disagreement about the relative rates of loss of the two types of product. In HeLa cells [179] apparently random excision of both products along both DNA strands occurred, but in mouse L cells, preferential loss of the diguanyl products with a half-time of 2 h has been claimed [174]. However, equitoxic doses of half-mustard gas and mustard gas had similar effects on synchronised HeLa cells, when dose~lependent delays in the cell cycle followed by rates of recovery to normal cell-cycle times were measured [179]. These data strongly suggest similar rates of recovery from mono- and bifunctional alkylation. This is difficult to reconcile with the reports of differential excision or a shorter half-time for removal of the diguanyl product. In bacteria, the ability to excise alkylation products from DNA is associated with increased resistance to the cytotoxic effects of mustards [128,129]. The situation in mammalian cells is less clear. Equal amounts of SM-induced repair replication and equal levels of DNA reaction were observed in 2 Yoshida sarcoma cell lines [201] which differ by a factor of 30 in sensitivity to the cytotoxic effects of SM. Some differences were found in the levels of HN2induced repair replication in sensitive and resistant lines and in one case a higher level of repair replication did correlate with decreased sensitivity [79]. Equal amounts of induced repair replication were observed in normal human fibroblasts and in xeroderm pigmentosum (XP) cells [46]. In all these cases, however, very high doses were used and only a single time point was measured, which introduces a number of problems of interpretation, i.e. repair may saturate at different doses in different cell lines and the time course may differ. That HN2-induced repair replication does saturate at high doses is illustrated by studies in human lymphocytes [45]. HN2 was shown to induce unscheduled DNA synthesis (UDS) in GI human lymphocytes using uptake of [SH]thymidine and subsequent liquid scintillation counting; the rate decreased over a 6-h period. Double reciprocal plots of rate of [3H]thymidine uptake against dose, after exposure to HN2 and other mutagens, indicated that different ratelimiting steps, and hence probably different enzymes, are involved in the repair of damage by MMS, HN2 and UV. The similarity between the time course of HN2- and UV-induced UDS led to the suggestion that common mechanisms were involved in repair of damage by these 2 agents. (For other evidence see section 6 on cross-sensitivity). In other studies, HN2~stimulated [SH]thymidine

138 incorporation into human l y m p h o c y t e s increased with increasing concentration (10-6--10 -4 M) then fell rapidly as the dose was further increased, probably due to inhibition of repair at high doses or cell killing [132]. That n o t all damage is excised before exponentially growing rodent cells reach their first post-treatment S phase is evident from observations on caffeine potentiation o f SM-induced cell lethality. The differential sensitivity of a pair of Yoshida sarcoma cell lines to SM-induced cell killing appears to be related to the existence, in resistant cells, of a caffeine-sensitive repair process absent in sensitive cells [149]. Resistant cells showed marked potentiation of SMinduced cell killing b u t no such potentiation was observed in sensitive cells [204]. No caffeine potentiation of SM- or HN2-induced cell lethality has been observed in human (HeLa) cells [177,230]. The actual mechanisms w h e r e b y inter- and intrastrand cross-links are removed from DNA are n o t completely clear. Removal of one arm of the crosslink would still leave an alkyl group attached to DNA which could inactivate the complementary strand with respect to providing a template for normally "error-free" excision repair unless a " c o p y choice" is involved. Simultaneous excision of both arms o f an interstrand cross-link would result in a doublestrand break which could be an inactivating lesion [132]. The whole question o f the relative proportions o f inter- versus intrastrand cross-links is still unresolved, b u t it still seems possible that only intrastrand cross-links can be removed, interstrand cross-links inevitably resulting in lethality. In assessing these data, particularly in relation to the use of cytotoxicityrelated assays in short term tests [29], it is important to bear in mind that measurement of c y t o t o x i c effects m a y give no real indication of mutagenic effects. The extrapolation from one to another must therefore only by undertaken with great caution. At best, the demonstration of a c y t o t o x i c effect can only give a qualitative indication that a c o m p o u n d is mutagenic.

6. Cross-sensitivity and resistance A knowledge of the cross-sensitivity patterns of different organisms to the inactivating and mutagenic effects of different classes of mutagens can be important when choosing appropriate organisms for use in test procedures. The response o f a variety of strains of bacteria and yeast to the c y t o t o x i c and mutagenic effects o f chemical mutagens is known to be under genetic control and evidence is accumulating that this is the case for mammalian cells from in vitro studies. Most of the data reviewed below relate to the c y t o t o x i c effects of nitrogen and sulphur mustard and the cross-sensitivity of mustard-sensitive strains to other DNA-damaging agents. There axe few data on the relative mutagenic effects of mustards, probably because they are poor mutagens even in strains selected for resistance to their c y t o t o x i c effects [138]. Evidence from a variety of eucaryotic and procaryotic micro-organisms suggests the involvement of similar pathways in the repair of UV- and mustardinduced damage. Strains o f bacteria [19,20,94,101,111], yeast [25,26], Neurospora [197] and slime moulds [169] which show sensitivity to UVinduced cell killing are frequently f o u n d to be cross-sensitive to HN2 inactiva-

139

tion but not to inactivation by X-rays and M M S . The enhanced sensitivityhas frequently been related to mutations in genes controlling excision repair. However, the existence of strains of yeast which are sensitive to X-rays and M M S as well as H N 2 and U V [26] suggests the additional involvement of genes controlling post-replication repair pathways and some interaction between the two pathways. Sensitivity to HN2-induced lethality in Drosophila embryos is associated with a mutation at the mei 6 locus which also confers ~/-ray, M M S and U V sensitivity [21,22]. The enhanced U V sensitivity is related to defective excision of pyrimidine dimers and with a defect in meiotic recombination [22]. Such observations again imply the involvement of both excision and recombination processes in the repair of U V and H N 2 damage. In mammalian cells, sensitivity to cell killing by H N 2 and S M is sometimes but not invariably associated with U V sensitivity. Yoshida sarcoma cells resistant to methylene dimethanesulphonate ( M D M S ) are cross-resistant to HN2, S M and U V [78]. However, a cell line resistant to M D M S but not to H N 2 has also been described [220]. Sensitive cells show reduced levels of UVinduced repair replication [79] and reduced capacity to excise dimers [171]. X-ray sensitive mouse l y m p h o m a L 5 1 7 8 Y cells show cross-sensitivity to UV, M M S , and E M S but in one case were not more sensitive to H N 2 than their more radioresistant derivative. A second pair of L 5 1 7 8 Y cell lines did show an approx. 2-fold difference in H N 2 sensitivity [54] and lines showing greater differences have also been described [88,90]. A HeLa cell line selected for M M S sensitivity showed enhanced sensitivity to HN2, UV, M M S and X-rays [159] thus mimicking the pattern in Drosophila [21,22]. However, a second clone selected for H N 2 sensitivity was not more sensitive than its parent line to M M S and X-rays [159]. Fanconi's anaemia cells are significantly more sensitive to cell killing by mitomycin C than HeLa or X P cells.This enhanced sensitivityis paralleled by increased frequencies of induced chromosome aberrations after H N 2 and mitomycin C exposure. Only moderate enhancement of UV-induced aberrations is observed [83,188,204]. The above data suggest that c o m m o n steps are involved in repair of UV-, HN2- and SM-induced damage in mammalian cells as in micro-organisms. However, the existence of mutants both of micro-organisms and mammalian cells which show sensitivity to HN2, UV, X-rays, and M M S indicates that considerable overlap in repair pathways m a y exist. Both excision and recombination pathways appear to be involved in repair of mustard-induced damage. There are, however, relatively few examples of pairs of mammalian cell lines which show differential sensitivity to HN2 or sulphur mustard where the level of reaction of the drug with DNA has been shown to be similar and hence where the differential sensitivity o f the 2 lines can be unambiguously related to differences in DNA repair. Many other changes in resistant cells have n o w been identified which suggest that patterns o f cross-resistance and sensitivity particularly in mammalian cells can be strongly influenced b y factors other than genes which control DNA repair. The acquisition of resistance to nitrogen mustard in mammalian cells is a multistep process suggesting that resistance is multifactorial [88]. Diverse biochemical features have n o w been identified which are associated with develop-

140 ment of resistance to HN2 in cultured cell lines. These include increases in celluar concentration of protective agents which spare critical targets, e.g. thiol groups [39,51,99,147], alterations in membrane permeability to drugs [188] and alterations in the efficiency of drug transport mechanisms [91,103,105, 139], all of which could result in lower levels of DNA reaction. As such, these alterations may confer resistance to the particular drug which is used to select the lines, e.g. HN2-resistant lines of L5178Y show an 18.5-fold decrease in sensitivity to HN2 but are only 2--3-fold more resistant that the sensitive line to other mustard derivatives, chlorambucil, melphalan and the cross-linking agent mitomycin C [91]. These other mechanisms, which are highly specific, may mask changes in cellular repair capacity. Hence, e.g. assay of nitrogen or sulphur mustard effects by cytotoxicity related assays in a permeability
Mutagenicity

Nitrogen and sulphur mustard and their derivatives have been shown to be mutagenic in a wide variety of species and much of the data has been extensively reviewed [ 7,127,138,228 ]. Table 3 lists the more recent observations and includes some earlier references to indicate the range of species tested. In most organisms these compounds have been shown to be mutagenic but there are some interesting exceptions which will be discussed below together with the possible mechanisms of induced mutagenesis where sufficient information is available. a. Micro-organisms

In phage, bifunctional alkylation is a predominantly lethal event. Concentrations of drug which induce a high level of kill are required to produce only a small increase in mutant frequency, i.e. nitrogen mustard is a very inefficient mutagen [53,134]. Both GC + AT transitions [53,134] and large deletions are produced, the latter predominating [ 53]. Deletions and base-pair substitutions [2,234] were also produced in bacteria [2,234]. Reversion of base-pair substitutions which require GC-~ AT transitions have also been reported in bacteria [37,47] and yeast [37,172,207]. The influence of repair genes on mutagenesis by a nitrogen mustard derivative [154] and a monofunctional mustard (2-chloroethyl ethyl sulphide, CEES) [84] has been studied. The repair defective E. coli strains exrA- uvrA- and recA- uvrB- were significantly more mutable than wild-type cells [84]. A recstrain of B. subtilis was significantly more sensitive to inactivation by a bifunctional mustard and was also more mutable [154]. Thus mustard-induced mutagenesis apparently does not depend upon the presence of recombination functions. Induction of mutations by CEES was also observed in exrA- strains which are non-inducible for SOS functions [84]. These data suggest that mutagenesis in repair defective strains may be the result of mis-replication across unexcised lesions (possibly O6-alkylated guanines) producing GC-> AT transitions. Wild-type strains were significantly less mutable, indicating that the pre-

D o s e - - r e s p o n s e c u r v e p l a t e a u s at ~ 2 5 0 ~ug/ml See t e x t

gal-chl d e l e t i o n s ; H I P A - r e s i s t a n t mutants

R e v e r s i o n o f b ase-palr s u b s t i t u t i o n s . no reversion of frameshift

Reversion of base-pair substitutions

No effect

Reversion of autoxotrophy

Reversion of auxotrophy

A u x o t x p h y e~g. a r g i n i n e r e q .

Proline independence, novobiocin resistance

S. t y p h i m u r i u m L T 2

S. t y p h i m u r i u m : his G 4 6 , his C 3 0 7 6

TA 1535

TA1537, TA1534, TA100

B. subtilis 1 4 9 r e c - h i s - i n d -

B. subtilis S B 2 5 rec+his-ind -

C o r y n e b a c t e r i u m sp. V U A 9 3 6 6

H a e m o p h i Z u s i n f l u e n z a e R d . p m B2

No i n d u c e d m u t a n t s r e c o v e r e d

Some m u t a g e n specificity claimed

V e r y l o w f r e q u e n c y in S B 2 5 r e c + strain

Dose r e s p o n s e a p p r o x i m a t e l y l i n e a r t o 250 #g/ml then plateau

I n c r e a s e in m u t a n t f r e q u e n c y w i t h i n c r e a s e in e x p o s u r e t i m e u p t o 3 0 rain. N o f u r t h e r i n c r e a s e at 9 0 m i n

Dose r e s p o n s e w a s l i n e a r in m u t a n t s which responded, recA-exrA-; uvrAuvrB-

A z e t i d i n e c a r b o x y l i c acid r e s i s t a n c e ; 5 - m e t h y l - t r y p t o p h a n resistance. D i r e c t m i s c o d i n g a n d possibly d e l e t i o n s

Dose r e s p o n s e a p p r o x , l i n e a r

t h r - --~ t h r ÷

F r e q u e n c y o f lac + r e v e r t a n t s 3 5 0 X spontaneous frequency after HN2 exposure, after MNNG 1000X.

32% of m u t a n t s a n a l y s e d w e r e s p o n t a n e o u s ; 22% o f i n d u c e d , GC -~ A T t r a n s i t i o n s , 47% d e l e t i o n s

W e a k m u t a g e n c o m p a r e d w i t h EMS b u t specificity similar

Remarks

E. coli B, B/r; K I 2 ; Bs__l; B s _ 2 ; w i t h m u t a t i o n s in v a r i o u s r e p a i r g e n e s

l a c - --* lac + r e v e r t a n t s ; forwal~t m u t a tions including deletions (non revertible) and probable base-pair substitutions (revertible)

R e v e r s i o n ; GC --~ A T t r a n s i t i o n s ; l a r g e deletions

No i n c r e a s e i n f o r w a r d m u t a t i o n b u t reversion of plaque-type RII mutants; GC --* A T t r a n s i t i o n s

Type of mutation

E. coil P 6 7 8

1I. Bacteria E. coli S

T 4 R I I intracellular

I. Phage T 4 in v i t r o

Organism

O R G A N I S M S IN WHICH THE M U T A G E N I C E F F E C T S OF N I T R O G E N AND S U L P H U R M U S T A R D H A V E BEEN S T U D I E D

TABLE 3

HN2

HN2

novembichin a

HN2

HN2

HN2

HN2

2-choroethyl ethyl sulphide

HN2

HN2

HN2

HN2

Agent

116

158

154

47

47

37

2

84

168

234

53

134

Reference

b.A

Recessive lethals; slow g r o w t h

P a r a m e c i u m aureUa

135 150a

H N 2 a n d a variety of derivatives

Loci affecting eye colour; deficiencies

Silkworm

136 HN2

HN2

D o m i n a n t a n d recessive lethals in F 1

M o r p h o l o g i c a l m u t a n t s i n scales

Habrobracon (oocytes)

66

3,6,7,8-1O, 36, 41, 67--72, 153, 212, 214

143, 144

83

140

1 5 5 - - 1 5 7 , 160

115

207,172

37

1 2 2 - - 1 2 4 , 165

15

117

Reference

E p h e s t i a kuhniella, larvae o f 1st i n s t a r

variety of HN2 derivatives

HN2

X-linked recessive l e t h a l s in s p e r m

Dose response linear w h e n plotted against m o l a r d r u g c o n c e n t r a t i o n

HN2 poor mutagen

HN2

SM

H N 2 a n d various derivatives

HN2

SM, H N 2 a n d derivatives

V a r i e t y o f loci

A r a b i d o p s i s thaliana

4X m o r e m o s a i c s t h a n c o m p l e t e s in F 1 endosperm

Nitrogen mustard was a poor mutagen relative to m o n o f u n c t i o n a l c o m p o u n d s

M a x i m u m m u t a t i o n yield achieved b y t r e a t m e n t a t o r n e a r a n d o f G1

Sex-linked recessive l e t h a l s ; deftciencies; a u t o s o m a l lethals; d o m i n a n t lethals; recessive visibles; m a j o r rearrangements

Colour and other m u t a t i o n s in endos p e r m . Partially fertility in F 1

Zea rnays (pollen)

V. I n s e c t s Drosophila m e l a n o g a s t e r

Chlorophyll mutations, wild-type to viridis o r a l b i n a i n M2 g e n e r a t i o n

T r i t i c u m a e s t i v u m (seeds)

Chlorophyll mutations, wild-type to viridis o r a l b i n a in M2 g e n e r a t i o n

R e v e r s i o n o f c y c 1-131; GC ~ A T transitions

Saccharo m y c e s cere visiae

I V . Plants H o r d e u m vulgate (seeds)

No r e v e r s i o n o f f r a m e s h i f t

S a c c h a r o m y c e s cerevisiae $ 1 3 8

HN2

HN2

Reversion of base-pair substitution

Saccharo m y c e s cerevisiae $ 2 1 1

M u t a g e n i c r e s p o n s e d i m i n i s h e s in strains h o m o z y g o u s f o r t a d 6 o r t a d 9

H N 2 a n d a variety of HN2 derivatives

HN2

R e v e r s i o n t o a~_inine i n d e p e n d e n c e

HN2

Agent

R e q u i r e m e n t for isoleucine or valine

w

Remarks

Aspergillus nidulans

Morphological mutants; reversion to adenine protxophy

Type of mutation

N. crasea inos 8 9 6 0 / a

I l l . F u n g i and p r o t o z o a Neurospora 38701

Organism

TABLE 3 (continued) to

ceils in vitro

I U d R s -* I U d R Z ;

hydrochloride.

Forward "mutation" T d R s -* T d R r

a Novembichin; 2-chloro-N,N-bis(2-chlozoethyl)propylamine

P388 mouse lymphoma

asn +

cells in vitro

L5178Y mouse lymphoma

am--*

Dominant lethals

a s h - -* asn÷

cells in vivo

Rat

L5178Y mouse lymphoma

VI. M a m m a l s a n d m a m m a l i a n cells in c u l t u r e Mouse Heritable partial sterility; recessive visible; dominant lethals

HN2

Dose response saturates at high doses; TdR data too limited

SM

Dose response linear

SM SM

-

SM, HN2, HN2n-oxide

--

-

--

4

40

40, 185

185

5, 8

f~

144

mutational lesion is uvr* excisable. The additional observation of reduced mutability relative to wild-type, in an endonuclease-II
b. Plants" Numerous nitrogen mustard and mustard gas derivatives have been tested for their mutagenic effects, e.g. induction of chlorophyll mutations in barley [160] and other plants (see Table 3 and [138] for references). In general the c o m p o u n d s are only weakly mutagenic probably because of their high toxicity. The effectiveness of HN2 as a mutagen in barley seeds has also been shown to be markedly d e p e n d e n t on temperature and pH of the treatment medium during exposure. Various other factors which can all be expected to influence the actual level o f reaction of mustards with DNA in critical sites have also been shown to influence m u t a n t yield, i.e. duration of exposure and concentration o f mutagen, size of seed and presence or absence of hulls [155--157]. Although chlorophyll mutations m a y well be of "point-mutational' origin, many of the other " m u t a t i o n a l " end points studied, e.g. reduction o f seedling growth, reduction of spike fertility [160] are probably more closely allied to chromosome breaking effects. Even in the well studied Tradescantia stamen hairs [215] it is n o t clear whether the mutational end points scored are the result of chromosome breakage followed b y deletion, chromosome non
145

c. Drosophila

In this species, both nitrogen and sulphur mustard have been shown to be active in inducing dominant lethal and visiblemutations, recessive sex-linked and autosomal lethals and visibles,as well as chromosomal mutations, i.e.deletions, inversions, duplications and translocations. Most stages of spermatogenesis are sensitive (see refs. 7 and 138 for reviews, and references in Table 3). A "storage effect" occurs in mustard-treated but not X-irradiated sperm when fertilisation is delayed. This results in a marked rise in translocation frequency but not in sex-linked recessive lethal frequency and has been extensively discussed by Auerbach [7 ]. Many derivatives of nitrogen and sulphur mustard have also been tested for mutagenicity in Drosophila and quantitative differences in the sensitivity of various spermatogenic cell stages reported [66,67,68,71]. N o qualitative differences in the types of damage induced were evident. In Drosophila as in micro-organisms there are a paucity of dose response data except for those of F a h m y and F a h m y [66] w h o observed a linearrelationship between the frequency of X-linked recessive lethals and molar dose for a variety of mustard derivatives in spermatozoa, and of Nasrat et al. [153] w h o quantitated other genetic effects of sulphur mustard by relating them to the frequency of X-linked recessive lethals (Fig. I). For mustard there is little indication of the "two-level effect" reported recently by Vogel and Leigh [224] for a variety of other compounds.

100

80

O O

i 60 |

A

0

A 40 A

o

20 A i £ 2

o

J 4

l 8

i 8

I 10

I 12

I 14

t 16

J 18

% sex iinked Recessive Lethals

Fig. 1. P r o p o r t i o n o f d o m i n a n t l e t h a l s t o s e x - l i n k e d recessive l e t h a l s i n d u c e d in s p e r m o f D r o s o p h i l a a f t e r e x p o s u r e t o X - r • y s , n; SM, o; MMS, m; a n d T E B , A. D • t • f o r X - r a y s a n d SM f r o m N a s r a t e t al. [ 1 5 3 ] , f o r MMS a n d T E B f~om V o g e l and L e i g h [ 2 2 4 ] .

146

Vogel and Leigh [224] analyse their data in terms of the ratio between the lowest effective concentration (LEC) required to produce a given genetic end point and the LDs0 dose, and conclude that the doses required to produce chromosome breakage effects such as dominant lethals, translocations and chromosome loss events are considerably higher than those required for the induction of recessive lethals or " p o i n t mutations". The data of Nasrat et al. [153] (Fig. 1), however, strongly indicate that dominant and recessive lethals, translocations and deletions, are induced over the same dose range, although their relative frequencies differ. We have been unable to find accurate LDs0 data for mustards in Drosophila b u t the fact that in a number of studies (Table 3) many different genetic effects were reported after exposure of flies to a single concentration, suggests that LDs0 : LEC ratios for various end points do n o t differ b y orders of magnitude. Superficially, therefore, these data suggest a fundamental difference in the m o d e of action of MMS and TEB as reported by Vogel et ai. and SM and related compounds. However, careful examination of their data suggests that expression of their results in terms of LEC : LDs0 ratios is potentially misleading since LEC has n o t in fact been accurately determined for end points other than sex-linked recessive lethals, e.g. LEC for MMS induced sex-linked recessives is quoted as between 1.0 X 10 -2 and 5 X 10 -2 mM and for dominant lethals 10-1--5 × 10 -1 mM. However, no dominant lethal frequency is reported for a concentration of 5 × 10 -2 M. Similar problems exist with respect to data for 2,4,6, triC1 PDMT in that no dominant lethal data are reported for the two lower concentrations (1 × 10 -3 mM and 3 × 10 -3 mM). We have plotted recessive lethal versus dominant lethal frequency for sulphur mustard and X-rays, and for MMS and TEB in Fig. 1. The data indicate no apparent differences between SM and the other two alkylating agents, with respect to these t w o genetic end points. d. Mammalian cells in vitro

Forward " m u t a t i o n " in P388 cells exposed in vitro to HN2 was measured as an increase in I U d R ~ or T d R ~ colonies [4]. The data indicate some dose-related increase in resistant colonies b u t the response flattens at the higher doses (Fig. 2a). Viability is low throughout the experiment at the higher doses (Fig. 2b) and expression curves show a marked peak at 48 h (Fig. 2c). It is now known that there are many problems associated with the use of antimetabolites as selective agents i n mammalian cells [80], in p a r t i c u l a r their selective stringency m a y be altered in cells suffering extensive alkylation. Other problems in the design of experiments to obtain accurate dose--response data have also been discussed [145]. These difficulties preclude at the present time interpretation of this dose--response relationship for HN2 mutagenesis. Limited data on the phenotypic stability and biochemical characterisation of some of these I U d R ~ and T d R ~ colonies are available [76,77] b u t the genetic lesion conferring resistance has n o t been identified. Thus, in view of the diverse origins of azaguanine-resistant phenotypes [80], the mutational origin of these clones should still be questioned. Exposure of L5178Y cell suspensions to SM in vitro resulted in a linear increase in reversion frequency from asparagine dependence to independence (asn- -~ asn ÷) with increasing dose [40]. Control reversion frequency in vitro

147

1001

b

1

!

60 50

I:

1

60

/

I

c

x

2[

t0 Z

I

"

I

0.04 0.08 0.16 Dose of HN2 I~M

,

20

,

,

,

40 60 80 100 ~me after HN2 treatment

,

120

F i g . 2. I n d u c t i o n o f I U d R r v a r i a n t s i n P 3 8 8 m o u s e l y m p h o m a cells in v i t r o . D a t a r e p l o t t e d f r o m A n d e r s o n a n d F o x [ 4 ] . (a) R e l a t i o n s h i p b e t w e e n i n d u c e d v a r i a n t f r e q u e n c y a n d d o s e o f H N 2 . e , f r e q u e n c y m e a s u r e d a t 4 8 h o n l y ; o, f r e q u e n c y m e a s u r e d a t 4 8 h a n d l a t e r , p o o l e d . (b) C h a n g e s i n v i a b i l i t y o f H N 2 - t r e a t e d P 3 8 8 cells w i t h t i m e a f t e r t r e a t m e n t w i t h v a r i o u s d o s e s o f d r u g . Cells w e r e e x p o s e d f o r 3 0 ' a t 3 7 ° C t o e , 0 . 1 4 p M ; o, 0 . 1 6 p M ; X, 0 . 3 2 ~ M ; t h e n c u l t u r e d f o r v a r i o u s t i m e s b e f o r e p l a t i n g a t 1 0 3 cells/ p l a t e i n t o F i s c h e r ' s m e d i u m s o l i d i f i e d w i t h s o f t a g a r . (e) C h a n g e s in H N 2 - i n d u c e d v a r i a n t f r e q u e n c i e s w i t h t i m e a f t e r e x p o s u r e t o v a r i o u s d o s e s o f H N 2 ; e , 0 . 0 4 ~ M ; G, 0 . 1 6 ~ M ; X, 0 . 3 2 ~M. T r e a t e d cells w e r e c u l t u r e d f o r v a r i o u s p e r i o d s o f t i m e in d r u g - f r e e m e d i u m b e f o r e p l a t i n g i n t o m e d i u m c n t a i n l n g I U d R f o r determination of variant frequency.

was 4.8 + 1.1 X 10 -7 which was increased to 7, 14 and 49 X 10 -6 by the increasing doses of SM (Fig. 3). A single injection of 100 /~g/kg SM of BDF1 mice inoculated intraperitoneally with L 5 1 7 8 Y asn- cells resulted in a 4-fold increase in reversion frequency when cells were subsequently assayed in vitro. The Do for SM-induced cell killing in L5178Y cells was 0.02/~g/ml, i.e. approx. 1 alkylation/1.5 × 106 nucleotides. Ten times more alkylations/g DNA are required to produce a similar increase in induced m u t a n t frequencies at several markers in E. coll. Although there is a lack of expression-time data, assays involving reversion from p r o t o t r o p h y to a u x o t r o p h y are probably free of many o f the problems discussed above; therefore these observations provide better evidence for mustard-induced mutation in mammalian cells. The asn--~ asn÷ marker also responded to agents which primarily induce base-pair substitutions

148

10

X - ray dose(fads) 100 1000 I

I

i

I

10-3

~

i0-4

10-6

0.01 0.1 1.0 Dose of Weogen or sulphur mustard(FM) Fig. 3. R e l a t i o n s h i p b e t w e e n i n d u c e d v a r i a n t o r m u t a n t f r e q u e n c y a n d d o s e , in m o u s e l y m p b o m a cells e x p o s e d t o X - r a y s , s u l p h u r m u s t a r d or H N 2 in v i t r o . A I U d R r i n d u c e d b y X - r a y s in P 3 8 8 l y m p h o m a cells in v i t r o . D a t a f r o m r e f . 7 6 . A T d R r i n d u c e d b y X - r a y s in P 3 8 8 l y m p h o m a cells in v i t r o . D a t a f r o m r e f . 76. o, H N 2 - i n d u c e d I U d R r v a r i a n t s in P S 8 8 l y m p h o m a cells. D a t a f r o m ref. 4, see also Fig. 2a. D a t a p o i n t s f r o m 4 8 h o n w a r d s p o o l e d , e , R e v e r s i o n f r o m a s n - --~ ash + i n d u c e d b y SM in m o u s e L 5 1 7 8 Y l y m p h o m a cells in v i t r o (see ref. 1 8 5 ) .

in procaryotes (EMS and MNNG) in a quantitatively similar way, and to the acridine half-mustards (ICR 372, ICR 191) which introduce primarily frameshift mutations. It is therefore not clear whether the change from asn- to asn÷ is due to a simple reversion or to a second forward mutation [218]. Attempts have also been made to demonstrate SM-induced reversion of asnL5178Y cells to asn÷ in vivo, by exposing mice, serially transplanted intraperitoneally with L5178Y cells, to vaporised SM for 6 h/day, 5 days/week over a 37-week period. The spontaneous frequency (asn- to asn ÷) in unexposed mice was 2.7--4.4/104 cells on in vitro plating [185]. There was no significant increase in frequency in exposed mice. However, these data can obviously not be interpreted as indicating a truly negative result as we have no indication as to the actual dose received by the target cells which were injected i.p.; the dose may be very low for this highly reactive compound. Assuming linear accumulation of dose with time, the authors calculate that "lung dose after 12 weeks exposure would be 0.29 mg/kg. This is approximately 3-fold higher than the i.p. dose of 100 ug/kg which Capizzi et al. used [40], and which resulted in a 4-fold increase in asn- -~ asn÷ reversion frequency. e. Germ cells o f mammals i. Antispermatogenic effects and dominant lethal induction in rats and mice. HN2 and several other aliphatic nitrogen mustards were tested for their effects on male rat fertility by sequential mating techniques [107]. Maximum tolerated doses of HN2 and isopropyl and n-butyl bis-(3-chloroethyl) amines

149

were administered i.p. and mating was continued for 12 weeks after treatment to sample all stages of spermatogenesis. Litter sizes did not differ from those in control matings over the whole 12-week period sampled. Other authors [86] have however reported antispermatogenic effects of aliphatic nitrogen mustards in rats; in this case animals given maximum tolerated doses, i.v., were sterile due to inhibition of spermatogenesis and testicular atrophy. Recovery eventually occurred, but only after 2--4 months. No information appears to be available on induction of dominant lethal mutations by nitrogen mustards in the rat but the relative resistance of the rat seminiferous epithelium to the cytotoxic effects of these compounds together with the lack of effects on subsequent litter size [107] suggests that dominant lethal effects may be minimal in this species. Route of injection may however be important since histological damage was reported after i.v. injection [86]. In mice, nitrogen mustards injected intraperitoneally produced testicular lesions [126]. Nitrogen mustard n-oxide induced dominant lethal mutations only in spermatozoa, with no effect on spermatids or spermatocytes, the dominant lethality being mainly due to post-implantation losses in matings 1--6 days after exposure to low dose levels (20 mg/kg) [60]. With increasing doses, post-implantation loss increased slightly and pre-implantation losses [1--6 days) increased drastically; there was no indication as to whether such early losses were due to failure of fertilisation, aspermia, or whether they represent a genuine increase in severity of effect on the genetic material [60]. This mustard derivative is however not thought to be itself an active alkylating agent, its possible metabolic pathways were described by Ishidate [106] and its distribution and excretion by Sator [191]. The metabolic products of this compound have been shown to react with proteins and nucleic acids, and it seems likely that the observed restriction of dominant lethal induction to spermatozoa is related to the distribution of enzymes required for its metabolic activation.

Dose of nitrogen mustard or nilxogenmustalxl n-oxide mg/kg

1.0

1.0

i0.0

,

i00

,

/

oI o / o

!

0.0 I0

, I 100 1000 X-ray dose (rads)

Fig. 4. I n d u c t i o n of d o m i n a n t l e t h a l s i n m o u s e s p e r m b y X-rays, o; n i t r o g e n m u s t a r d , X; a n d n i t r o g e n m u s t a r d n - o x i d e , o. D o m i n a n t l e t h a l f r e q u e n c y is e x p r e s s e d as 1 m i n u s live e m b r y o s / i m p l a n t s f o r t r e a t e d m a l e s d i v i d e d b y live e m b r y o s / i m p l a n t s i n c o n t r o l s . X-ray d a t a f r o m ref. 221 ; n i t r o g e n m u s t a r d d a t a r e c a l c u l a t e d f r o m r a w d a t a k i n d l y s u p p l i e d b y Dr. D. A n d e r s o n ; d a t a for n i t r o g e n m u s t a r d n - o x i d e f r o m ref. 60.

150 HN2 was used as a "positive mutagen c o n t r o l " in inter-laboratory studies on dominant lethal induction in male mice [5]. At the 2 dose levels used (2.5 and 3.5 mg/kg) a fall in total implants/pregnancy and a slight rise in early deaths/ pregnancy was evident in the first 3 post~treatment weeks. There was little indication of a dose--response relationship (Fig. 4). The effects of HN2 apparently extend in time b e y o n d those of nitrogen mustard-n-oxide which m a y be related to differences in the relative distribution and pharmacokinetics of the 2 compounds. Negative results in dominant lethal tests with HN2 in mice have been reported b y other workers [43,63]. Dominant lethal induction has been studied in male rats chronically exposed to SM vapour [185]. Two doses were used and exposure was for 1--52 weeks. No significant increase over control values was evident in animals exposed to the lower dose 0.001 mg/m 3. The incidence was 3.96% + 1.9 in controls and 4.4% + 1.1 in exposed group. Significant dominant lethal effects were seen at the higher dose (0.1 mg/m3). There was a cumulative increase with duration of exposure which reached 9.4% ± 1.9 at 12 weeks (total dose inhaled 0.63 mg/kg). The most sensitive cells were the spermatozoa which is consistent with the probable lack of repair systems in this differentiated cell t y p e [202]. ii. Induction o f translocations and recessive visibles in mice. Falconer et al. [73] treated male mice of the CBA strain with nitrogen mustard at dose levels close to the LDs0. The males which survived were initially fertile for 0--8 days, subsequently became sterile for 26--30 days, then fertility returned. The F~ progeny of the treated males was examined for the presence of visible recessive mutations and heritable semi-sterility due to translocations. A total of 67 progeny were produced during the early fertile period and 82 after fertility returned. No dominant mutations or genetic loss at 5 marked loci were detected. One recessive visible, "crinkled", was recovered. 71 F1 males were tested for fertility and amongst them 2 completely sterile males, 2 semi-sterile and 3 suspected semi-steriles were detected. 1 of the 2 semi-sterile males was analysed genetically and cytologically and was found to carry 2 independent translocations. The authors conclude that at least 1 of these translocations must have arisen de novo in treated sperm b u t the relationship between the origin of the visible mutation and treatment could n o t be proven. To our knowledge no specific locus data are available. It is generally accepted that chromosome breakage is responsible ~for dominant lethal induction; thus the apparent differences in duration of the dominant lethal effect observed for the different nitrogen mustard derivatves may be directly related to the actual levels of DNA alkylation achieved in the sensitive target cells. Although no data appear to be available on DNA alkylation levels or on the ability of mammalian germ cells to repair damage induced by HN2 and SM there is a considerable literature on their ability to repair damage induced by alkanesulphonic esters [202]. The DNA of late spermatids and mature spermatozoa were extensively alkylated. These stages gave rise to EMS-induced dominant lethal mutations and do n o t appear to be capable of repairing DNA lesions. Earlier stages of spermatogenesis, mid and early spermatids, did show repair as measured b y incorporation of [3H]thymidine. Dose-response curves for induction of UDS were linearly related to administered dose. The lowest injected dose which caused a measurable induction of DNA

151 repair was from 5 to 15 times lower than that required for induction of dominant lethals and translocations in those cell stages in which repair can be elicited. Although in some cases, therefore, there may be a correlation between inability of cell stages to undergo DNA repair and their sensitivity to dominant lethal induction, the correlation is not absolute as EMS induces dominant lethals in early spermatids which are proficient in DNA repair. Lack of genetic effects in certain germ cell stages may well be due to efficient repair of induced damage or selection against heavily damaged cells or lack of induction of damage for pharmacokinetic reasons. 8. Cytogenetically detected effects The first demonstration of the induction of chromosomal translocations by SM was in Drosophila [9] and shortly afterwards observations of other chromosome structural aberrations, chromosome stickiness and defects in anaphase segregation of chromosomes were made in plant cells treated with this agent [56]. These phenomena, particularly structural damage (i.e. chromosome breakage or clastogenesis) have now been detected in a wide variety of plant and animal cells exposed to nitrogen and sulphur mustard (Table 4). a. Clastogenesis i. Types o f aberration. HN2 and SM induce all the various types of chromatid aberrations (i.e. gaps, deletions and exchanges, see Savage [192] for classification) which are observed in cells exposed to ionising radiation [56,75, 205] though not in the same proportions or as randomly distributed within and between chromosomes [75,219]. The mustards induce few, if any, true chromosome-type aberrations [65,75,200,205] which are characteristic of cells X-irradiated in GI [64]; those which are observed are probably derived from chromatid-type aberrations [198]. In addition to conventional chromosomal aberrations the mustards have been reported to induce chromosome "shattering" or gross fragmentation, particularly at high doses [57,58,121]. ii. Lesions and mechanisms involved in aberration formation. It has long been suggested that DNA cross-links are the lesions responsible for chromosomal aberrations in cells treated with polyfunctional alkylating agents including the mustards [87] although it is clear that monofunctional agents (e.g. a monofunctional mustard [156]) are also clastogenic but usually only at considerably higher doses [138]. Conventional aberrations induced by HN2 and SM are formed at the time of DNA synthesis, presumably as replication errors at the sites of alkylation of parental DNA [65,205]. If interstrand cross-links are the lesions responsible for aberration induction we might expect the aberrations to affect both sister chromatids at identical loci after semi-conservative replication [65]. However, apart from isochromatid deletions, the mustards induce aberrations which affect only one of the two sister chromatids which suggests that intrastrand cross-links [74,130] may be resposible [201]. The frequency of chromosomal aberrations in cells treated with mustards will depend upon the initial extent of alkylation, the amount of repair/removal of lesions before cells undergo DNA synthesis and the probability of persisting

G e r m cells,

in vivo

in vivo

SM

Drosophila melanogaster

Sperm

Ascites tumours Carcinoma Sarcoma

HN2 HN2 HN2 SM HN2 SM HN2 HN2 HN2 HN2 HN2

Mouse Chinese hamster Chinese hamster Potorous tridaetylus Mouse Rat Rat Mouse Mouse Rat Mouse

HN2 HN2 HN2 HN2

HN2 SM SM

HN2 and derivs HN2

SM HN2 HN2 HN2 HN2 HN2 HN2

Agent

Skin Ovary Lung Lymphocytes Sarcoma Lymphosarcoma Bone marrow

Human

Lymphocytes

in vivo

Non-human S o m a t i c ceils, i n v i t r o

Human

Lilium lancifolium T r a d e s c a n t i a bracteata Tradescantia b m e t e a t a

Crepis capillaris

Lymphocytes

P o l l e n m o t h e r ceils P o l l e n m o t h e r cells Pollen grains

H o r d e u m vulgare

Allium flstulosum H o r d e u m vulgate Lilium henry i Vicia f a b a

A l l i u m cepa

Species

B. A n i m a l Human S o m a t i c cells, i n v i t r o

G e r m cells

Root tips

A. Plant S o m a t i c cells

Shoot tips from treated seeds

S p e c i f i c cell t y p e

C l a s ~ f i e a t i o n o f cell t y p e

CYTOGENIC EFFECTS OF NITROGEN AND SULPHUR MUSTARD

TABLE 4

stickiness stickiness

stickiness

polarity defects

Structural

Structural Structural, SCE Structural Structural Structural Structural Structural Structural Structural Structural Structural

Structural Structural, numerical Structural Structural, numerical

Structural, polarity defects, stickiness Structural, polarity defects stickiness Structural, polarity defects, stickiness

Structural

Structural

Structural, Structural Structural, Structural Structural, Structural, Structural

Effect

9,210

16 100,170 217 2M 16 199,201 16,57 231 196,231 120.121 196,73

189 152 205, 193 48,152

190 56 56

11

56 16 162 12, 58 219 162 65, 75, 112, 113, 173, 175, 217 156

Reference

¢~ t~

153 lesions giving rise to replication errors in the form of aberrations. Pre-replication repair can be in the form of excision of alkylated bases [177,180--182] or the "unhooking" of one arm of a cross-link [174,233]. The probability of persisting lesions giving aberrations appears to depend upon the capacity of the treated cell for post-replication repair, i.e. the "filling in" of gaps formed in newly synthesised DNA opposite lesions in parental DNA [38,131], unrepaired gaps leading to aberrations [114,177,199].

iii. Dose--response relationships. Studies on the clastogenicity of mustards have been performed with only 1 or 2 doses and/or with limited sampling of asynchronous populations of cells from which the shapes of dose--response curves are impossible to interpret [138]. A multidose study on a synchronous population of cells is required to obtain meaningful dose--response data. iv. Human cells. Data on clastogenicity of mustards on human cells in vivo are very limited (Table 4). Cohen and Lansky [48] found 4.4% of lymphocytes with chromosomal aberrations in blood cultures set up 4 days after treatment of a cancer patient with 12 mg HN2; this is about the amount of damage expected from exposure of human lymphocytes to 40 fads of X-rays (250 kV, 100 rads/min [133]) although the spectrum of aberration types is different. Confidence limits (95%) on this HN2-induced aberration frequency were 1.2 to 7.6%, equivalent to approximately 2 and 70 fads respectively by extrapolation and interpolation. Even this rough estimate of the "tad equivalent chemical" (REC, [27] ) is subject to several assumptions whose validity may be uncertain, i.e.: (1) that the level of HN2 damage in cells at 4 days after treatment is the same as that at the time of treatment; (2) that the use of a 72-h culturing time g~ves a correct estimate of the amount of chromosome damage induced in G0 lymphocytes by HN2; (3) that the X-ray sensitivity of human lymphocytes is similar in vivo and in vitro, since the tad equivalents given above were derived from in vitro data. Equality of in vivo and in vitro sensitivity is probably a correct assumption (references in [14]); (4) no error on the dose--response data for X-ray induced damage. Sharpe [205] analysed 100 lymphocyte metaphases from a patient with Hodgkin's disease before, and at 24.5 and 41.5 h after treatment with a single dose of 15 mg HN2 and observed zero, two and zero aberrations respectively, excluding a few gaps. The 2% yield at 24.5 h is formally equivalent to an X-ray dose of about 10 rads [133] with the 95% confidence limits on the aberration yield (+2.7%) giving rad equivalents of between 0 and 35 fads. Nasjletti and Spencer [152] observed increases in aberrration frequencies of up to 21% above pre-treatment levels during HN2 therapy for bronchogenic carcinoma but do not state the dose given. Significantly they noted a decline in aberration frequencies after therapy, with no detectable chromosome damage 3 months after treatment. This will result at least partly from the repair of pre-aberration lesions before DNA synthesis which occurs in vitro under the stimulus of PHA (see section 8a, ii). Clearly, data on the in vivo sensitivity of human lymphocyte chromosomes to HN2 are subject to large uncertainties and even if accurate data were available, it is doubtful if meaningful extrapolation to germ cells could be made [213].

154

TABLE 5 C H R O M O S O M E N U M B E R S O F H U M A N P E R I P H E R A L B L O O D L Y M P H O C Y T E S T R E A T E D I N VIT R O W I T H H N 2 (Ladner and S c o t t , u n p u b l i s h e d )

H o u r s after treatment

24

48

Concentration (/~g/ml)

0 0.02 0.10 0.20 0 0.02 0.10

% Cells w i t h f o l l o w i n g c h r o m o s o m e Nos. 44

45

46

47

4

2 12 10 12

96 86 86 70

2 2 4 12

6 16 14

92 78 84

2

14

80

2

4

0.20

48

P o l y p l o i d cells n o t c o u n t e d .

b. Sister-chromatid exchanges (SCEs) SCEs can be induced by HN2 at considerably lower doses (3 X 1 0 -7 M in cultured Chinese hamster cells) than are required for clastogenesis [170]. However, it has not yet been shown that SCEs represent mutagenic events. c. Chromosome stickiness, segregational errors and numerical abnormalities The mustards have been found to induce chromosome "stickiness" in meiotic and nitotic cells [56,190,219] and to interfere with the normal segregation of chromosomes at anaphase [56,121,190]. Both these effects are possible candidates for producing chromosome numerical changes, i.e. polyploidy or aneuploidy, the latter being particularly relevant to genetic ill-health in man [42]. Although there are no reports of induced aneuploidy in meiotic cells treated with mustards, it is of interest that polyploidy and aneuploidy were observed in lymphocytes from the 2 HN2-treated cancer patients investigated

TABLE 6 LDs0 V A L U E S F O R V A R I O U S R O U T E S OF I N J E C T I O N IN R O D E N T S Drug

Route of injection a

Species

LD50 dose

References

HN2

s.c. orally i.p. i.v. i.p. i.p. i.p.

mouse mouse mouse rat rat rat rat (litter L D s 0 ) t e r a t o g e n i e range

1-- 4 mg/kg 10--20 mg/kg 4.4 m g / k g 1.1 m g / k g 2.5 m g / k g 1.8 m g / k g 2.0 m g / k g 0.6--1.0 mg/kg

102 23 216a 216 149 216a 149 149

SM

i.v.

rat

nitrogen mustazd n-oxide

i.v. i.D.

rat mouse

a s.c., s u b c u t a n e o u s ; i.v., i n t r a v e n o u s ; i.p., intraperitoneal.

0.7 m g / k g

216

60.0 mgfkg ~80.0 mg/kg

195 60

155 by Nasjletti and Spencer [152] and also after their in vitro HN2 treatments. Similarly, Conen and Lansky [48] observed aneuploid lymphocytes from a cancer patient on HN2 therapy. In this laboratory we have also observed HN2induced aneuploidy in human lymphocytes treated after 48 h in culture {Table 5). On the basis o f these findings in somatic/mitotic cells it would seem worthwhile to investigate the possibility of mustard-induced aneuploidy in meiotic cells of laboratory mammals with a view to assessing the possible risks in man. 9. Acute toxicity Some LDs0 values for mustards are summarised in Table 6. Rodents given single near LDs0 doses, die between 4 and 14 days after injection [216a]. The cause of death can be attributed to the cytotoxic effects of these compounds on rapidly renewing normal tissues including lymphoid tissues, stomach, small and large intestine and bone marrow [62,216a]. Profound pathological changes in all these tissues have been described. At supralethal doses paralytic and cholinergic effects are seen. In man the major toxic effect o f therapeutic doses of HN2 is depression of normal haematopoietic function. 10. Carcinogenicity The available data on carcinogenicity of a variety of mustards have been collected in an IARC monograph on the topic [102] and therefore will not be considered in detail here. Carcinogenicity tests have been performed in both rats and mice and a variety of routes of administration have been used. Data TABLE 7 CARCINOGENECITY Total dose Nitrogen mustard

50 mg/kg 0.4 mg/kg 2.0 mg/kg 4.0 mg/kg 3.3 mg/kg 6.64 mg/kg 9.6 mg/kg 0.038 mg/kg 0.144 mg/kg 0.85 mg/kg 3.36 mg/kg

O F M U S T A R D S IN R A T S A N D M I C E

Schedule --

mo

Route a

Site o f t u m o u r

References

s.c. i.v. i.v. i.v. i.v. s.c. i.v. i.p. i.p. i.p. i.p.

l u n g , liver, u t e r u s lung lung lung lung skin p a p i l l o m a s , l u n g thymic lymphoma lung lung lung lung

i.v.

t u r n o u t s in a v a r i e t y o f organs

s.c. i.v.

skin p a p i l l o m a s lung

97 98

inhalation

lung

98

use

1 X 1 #g/week single d o s e 2 X 1 mg 2 d interval 2 X 2 mg 2 d interval 1 2 X 0 . 2 8 m g in 4 w e e k s 8 X 0.83 mg weekly 4 X 2.4 mg at 2-week intervals 12 × 0.0031 mg 12 × 0.012 mg 12 × 0.071 mg 12 X 0.28 mg over 4 weeks

23 96

97 59 208

N i t r o g e n m u s t a r d - - rat

5.72 mg/kg Sulphur

mustard

4.15 mg/kg 0.252 mg/kg Total dose unknown

52 × 0.11 mg weekly --

mo

195

u se

5 X 0.83 weekly 4 X 0.063 mg every 2 d

a s.c., s u b c u t a n e o u s ; i.v., i n t r a v e n o u s ; i.p., i n t r a p e r i t o n e a l .

156 are summarised in Table 7. Life shortening effects of fractionated and acute dosage of female RF mice with HN2 were reported by Conklin et al. [49,50]. The life-shortening effect was not entirely attributable to the induction of neoplasia or any one particular group of effects, but rather was related to the premature development of many diseases of old age. In these experiments HN2 treatment also resulted in an increased incidence of tumours at all sites, e.g. thymic lymphomas and lung tumours, and although the frequency of other tumours, e.g. myeloid and other leukaemias and ovarian turnouts was not increased, they occurred earlier than in untreated animals [50] in all cases. The available evidence [161,225] (see also IARC Monograph [102]) also suggests a strong correlation between cancers of the upper respiratory tract and levels of individual exposure to mustard gas (SM) during World War I [235]. Exposure during manufacture of SM, which continued up to 1945, also resulted in increased tumour incidence. Thus nitrogen and sulphur mustards are undoubtedly carcinogenic in experimental animals, mainly rodents, inducing pulmonary turnouts and in some instances sarcomas, lymphomas and leukaemias. Sulphur mustard is carcinogenic in man. Nitrogen mustard is used in the MOPP regime (nitrogen mustard, vincristine, prednisolone and procarbazine) for the treatment of Hodgkin's disease, and a number of such patients have developed second neoplasms, mainly acute myeloid leukemia (AML). Data for many of these patients are available only in single case reports and it is difficult to estimate an overall frequency; however, there exist several surveys from which it is possible to make some estimates of frequency since the number of patients observed, the length of time over which the observations were made and the number of patients in which AML developed is specified [206]. The average incidence of AML in the U.S.A. population is 2.2/100 000/year; in most of the surveys of Hodgkin's patients the incidence ranged from 15--25/ 100 000/year and in 2, from 156/100 000/year to 406/100 000/year. The 2 latter surveys however, come from small groups of patients treated relatively recently. A similar picture emerged from surveys of the incidence of AML in myeloma patients and in 6 surveys the incidence ranged from 20--500/ 100 000/year. The shortcomings of these estimates have been discussed [216] and they may be biased since the cases in which AML does develop are more likely to be reported than those in which it does not. In addition, patients suffering from such malignancies may have an inherent predisposition to second tumours. However, in view of the known carcinogenicity and mutagenicity of nitrogen mustard in animals the evidence is highly suggestive. The benefit of such treatment in malignant disease is not generally disputed. The issues become more complex when nitrogen mustard derivatives such as melphalan and cyclophosphamide are used to treat patients with non-malignant disease such as rheumatoid arthritis, psoriasis and chronic renal disease and as immunosuppressives in renal transplantation. Approximate rad~quivalent values [27] can be calculated for therapeutic exposure to the MOPP regime by comparison of increased AML incidence amongst patients, with AML incidence amongst A-bomb survivors. To assess the precise contribution of nitrogen mustard to this increase is however impossible since the mutagenic effects of all other components and their possible interactions when used in combination are not known.

157 11. Teratogenicity Teratogenicity of nitrogen mustard was first demonstrated in amphibian embryos in the late 1940s [17]. Subsequently, teratogenic effects in rats [93] and mice were reported [55]. In mice [55] small doses (1--2 lzg, i.p.) on days 11--12 of pregnancy resulted in foetal abnormalities without noticeable maternal toxicity; higher doses produced lethal effects on all embryos. In rats [93] 0.5 mg/kg between days 10--16 of gestation produced a range of foetal abnormalities (see Table 6 for LDs0 values). Injection of rats on day 12 of pregnancy has produced the most consistent teratogenic effects [148--150]; 100% of litters were resorbed after a dose of 1.4 mg/kg (maternal LDs0, 1.75 mg/kg) and litters were 100% normal after 0.35 mg/kg. Doses between 0.35 mg/kg and 0.7 mg/kg produced a range of foetal abnormalities amongst liveborn including syndactyly, encephalocoele and cleft palate. Chlorambucfl tested in parallel [150] produced similar effects although the dose required was higher (litter LDs0, 10 mg/kg), presumably a reflection of the need for metabolism of this compound. Both nitrogen mustard and chlorambucil given earlier than day 12 of pregnancy were more embryotoxic [30,150]. Inhalation of vaporised sulphur mustard (0.1 mg/m 3) by pregnant rats was without effect on embryonic mortality and failed to cause a significant increase in gross foetal abnormalities [185]. Teratogenic effects in chick embryos have also been studied [149]; HN2 was injected into the yolk sac of 4~lay embryos which were sacrificed at 18 days. The LDs0 for HN2, as measured by failure to hatch, is 0.01 mg/egg. Marginal teratogenicity was reported. 12. Germ.cell hazard: problems o f extrapolation to man

Attempts have been made to estimate risk of exposure to several cancer chemotherapeutic agents, e.g. cyclophosphamide and myleran using specific locus and translocation data from the mouse [95]. To our knowledge, no specific locus data are available for either nitrogen or sulphur mustard and only limited translocation data [75]. It is, therefore, not possible to make similar estimates for these compounds. Rad-equivalents have been used [27] in attempts to quantitate the risks of chemical exposure relative to radiation exposure. Considerable difficulties also exist with this approach as often the actual dose of chemical received by the target tissue is not known and the value obtained depends on the end point used. We have attempted to calculate rad-equivalents for nitrogen mustard exposure, but in most cases the data are too limited for such calculations to be meaningful. This problem is reflected in the wide variation in values obtained (Table 8). The problems inherent in using chromosomal aberrations in human lymphocytes have already been pointed out (see Section 8). Difficulties with respect to the data for induced "mutation" in mammalian cells in vitro (P388) have also been discussed (Section 7d). The available dose--response data for induced "mutation" in P388 cells for X-rays and nitrogen mustard are plotted in Fig. 3. Completely different rad~equivalent values are obtained when calculations are based on the "mutation frequency" to thymidine resistance [76] (450 fads) as opposed to IUdR resistance (100 fads).

Chromosome structural aberrations

Chromosome structural aberrations

IUdR r TdR r

D o m i n a n t lethals Early deaths

T r a n s l o c a t i o n s , i n d u c e d semi-sterility

I n c r e a s e in i n c i d e n c e o f A M L

H u m a n l y m p h o c y t e s in vivo and in vitro

H u m a n l y m p h o c y t e s in vivo a n d in vitro

P 3 8 8 l y m p h o m a in v i t r o

Mouse s p e r m a t o z o a

M o u s e p o s t - m e i o t i c g e r m cells

Man

a A c t u a l dosage n o t specified b u t at least 6 × 0 . 1 5 m g / k g .

End point

Cell o r o r g a n i s m

R A D - E Q U I V A L E N T V A L U E S FOR V A R I O U S END POINTS

TABLE 8

MOPP a regime

a p p r o x . 3.0 m g / k g

2.5 m g / k g 3.5 m g / k g

2.25/~g/ml

15 mg to patient

12 mg to patient

Dose of mustard (HN2)

150/105

3--5% o f o fspring

14.7% 12.9%

2.7 × 1 0 - 3 m u t a n t s / s u r v i v o r 2.7 × 1 0 -3 m u t a n t s / s u r v i v o r

2.0 +- 2.7% a b e r r a t i o n s

4.4% a b e r r a t i o n s ( c o n f i d e n c e limits 1 . 2 - - 7 . 6 % )

Amount o f induced damage

approx. 400

a p p r o x . 80

approx. 150

approx. 100 approx. 450

10 ( 0 - - 3 5 )

40 ( 2 - - 7 0 )

Rad-equiva l e n t (rod)

206,221

73, 221

5, 2 2 1

4 75

205,133

4 8 , 133

References

00

159

o

40

30

| g "~ 20

.

0 me~e'- I

I 200

1

""

.

I I 4.00 X-ray dose (rads)

I 600

I

I 800

Fig. 5. R e l a t i o n s h i p b e t w e e n t r a n s l o c a t i o n f r e q u e n c y in s p e r m a t o g o n i a (o) a n d p o s t - m e i o t i c g e r m cell stages (o) o f t h e male m o u s e a n d X-ray dose. D a t a f r o m ref. 2 2 1 .

Difficulties also exist with respect to mouse dominant lethal data [5] which are so limited (Fig. 4) that it is even difficult to assume a dose--response relationship. However, 2.5 mg/kg could be considered to be very approximately equivalent to 140 fads. The value given for translocations in the mouse is based on the observation of Falconer et al. [73] of two sterile males and two semisterile males amongst the progeny of nitrogen mustard treated males; a maximum translocation frequency of 5.5%. A very approximate rad~equivalent of 80 fads is obtained on comparison with the also limited data for X-ray induced translocations in post-meiotic germ cells of the mouse (Fig. 5). We conclude that it is impossible at the present time to make any meaninggul estimate of the potential genetic hazard of mustards to man. 13. Conclusions Both nitrogen and sulphur mustard are mutagenic in a wide variety of species; there are however a few notable exceptions mainly amongst microorganisms. Mutation in rec- strains of E. coil and B. subtilis appears to be the result of direct miscoding on replication across alkylated sites (possibly O 6alkylated guanines) resulting in G-C-~ A-T transitions. In wild type E. coli, mutation is more likely to result from "error-prone" repair. Both compounds produce deletions in micro~)rganisms and chromosome (including deletions) structural aberrations in a wide variety of plant and animal material including Drosophila. Nitrogen mustard has also been shown to produce sister-chromatid exchanges. Cytotoxicity in micro-organisms and cultured mammalian cells appears largely to result from unrepaired DNA cross-links which constitute a complete block to DNA replication. Both nitrogen and sulphur mustard are teratogenic and carcinogenic in rodents and sulphur mustard is known to he carcinogenic in man. The many derivatives of both compounds appear to differ only quantitatively in their mode of action. Despite the extensive literature, the

160 lack of dose--response data particularly in rodents makes if difficult to assess the risk of genetic damage in man. Acknowledgements The authors would like to thank Mr. John Wasson, Environmental Mutagen Centre, Oak Ridge, TN, U.S.A., for supplying a print~out o f relevant literature and numerous reprints o f publications. They would also like to thank Dr. B.W. Fox for help with the section of "Chemistry and overall levels of DNA alkylation". References 1 A d a i r , F . E . , a n d H . J . Bagg, E x p e r i m e n t a l a n d clinical studies o n t h e t r e a t m e n t of c a n c e r b y d i c h l o r o e t h y l s u l p h i d e , A n n . Surg., 9 3 ( 1 9 3 1 ) 1 9 0 - - 1 9 9 . 2 A l p e r , M., a n d B.N. A m e s , Positive s e l e c t i o n o f m u t a n t s w i t h d e l e t i o n o f t h e gal. chl r e g i o n o f t h e S a l m o n e l l a c h r o m o s o m e as a s c r e e n i n g p r o c e d u r e f o r m u t a g e n s t h a t cause deletions, J. 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