Genetic effects of dimethyl sulfate, diethyl sulfate, and related compounds

63

Mutation Research, 75 ( 1 9 8 0 ) 6 3 - - 1 2 9 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press

GENETIC EFFECTS OF DIMETHYL SULFATE, DIETHYL SULFATE, AND RELATED COMPOUNDS

G E O R G E R. H O F F M A N N *

Department of Biology, Meredith College, Raleigh, NC 2 7611 (U.S.A.) ( R e c e i v e d 5 April 1979) ( A c c e p t e d 5 July 1979)

Table o f contents Summary ................................................... Introduction ................................................. Chemical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Toxicology Acute toxicity .......................................... Carcinogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transplacental effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interaction with DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R e p a i r processes R e p a i r o f alky]ated D N A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E r r o r - p r o n e repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Overview o f m u t a g e n i c i t y d a t a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G e n e t i c e f f e c t s in viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G e n e t i c e f f e c t s in p r o k a r y o t e s I n d u c t i o n o f base-pair s u b s t i t u t i o n s in bacteria . . . . . . . . . . . . . . . . . . . . . I n d u c t i o n o f f r a m e s h i f t m u t a t i o n s in bacteria . . . . . . . . . . . . . . . . . . . . . I n d u c t i o n o f d e l e t i o n s in bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . O t h e r m u t a t i o n a l s t u d i e s in bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . M u t a g e n i c i t y in t r a n s f o r m i n g D N A . . . . . . . . . . . . . . . . . . . . . . . . . . . . M u t a g e n i c i t y in blue-green algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R e c o m b i n o g e n i c i t y in bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bacterial repair assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prophage induction ....................................... G e n e t i c e f f e c t s in fungi Induction of mutations .................................... Induction of mitotic recombination ............................

64 64 65 68 68 70 70 77 78 79 80 89 92 93 93 95 96 96 96 97 98 101

* Present address: Board on Toxicology and Environmental Health Hazards, National Academy of Sciences, 2101 Constitution Avenue NW, Washington, D.C. 20418 (U.S.A.).

Abbreviations: BSF, 103-butylene sulfate; DES, diethyl sulfate; DMS, dimethyl sulfate; DMSO° dimethylsulfoxide; DPS, dipropyl sulfate; EMS0 ethyl methanesulfonate; ENU, N-ethyl-N-nitrosourea; ESF, 1,2-ethylene sulfate; iPMS, isopropyl methanesulfonate; MMS, methyl methanesulfohate; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; MNU, N-methyl-N-nitrosourea; PSF, 1,3-propylene sulfate; TMV, tobacco mosaic virus; XP, xeroderma pigmentosum.

64 Genetic effects in vascular plants Induction of mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Induction of chromosomal alterations . . . . . . . . . . . . . . . . . . . . . . . . . . Induction of paramutational changes . . . . . . . . . . . . . . . . . . . . . . . . . . . Factors modifying response to mutagens . . . . . . . . . . . . . . . . . . . . . . . . . Possible interactions among mutagens . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic effects in insects Induction of mutations in Drosophila . . . . . . . . . . . . . . . . . . . . . . . . . . . Induction of male crossing-over in Drosophila . . . . . . . . . . . . . . . . . . . . . Induction of chromosome rearrangements in Drosophila . . . . . . . . . . . . . . Studies with other insects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic effects in fish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic effects in mammalian cells in culture Induction of mutations and chromosome aberrations . . . . . . . . . . . . . . . . Induction of sister-chromatid exchanges and unscheduled DNA synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic effects in mammals Induction of dominant lethals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Induction of cytogenetic alterations . . . . . . . . . . . . . . . . . . . . . . . . . . . . Induction of specific-locus mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . Inhibition of testicular DNA synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . Note added in proof . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

102 105 106 106 107 109 111 111 112 113 113 113 114 115 116 117 117 117 117

Summary DMS and D E S are m o n o f u n c t i o n a l a l k y l a t i n g agents t h a t have been s h o w n t o i n d u c e m u t a t i o n s , c h r o m o s o m a l aberrations, a n d o t h e r genetic alterations in a diversity o f organisms. T h e y have also been s h o w n t o be c a r c i n o g e n i c in animals. As an a l k y l a t i n g agent, DMS is a t y p i c a l SN2 agent, a t t a c k i n g p r e d o m i n a n t l y n i t r o g e n sites in nucleic acids. D E S is capable o f SN1 a l k y l a t i o n s as well as SN2 a n d t h e r e b y causes s o m e a l k y l a t i o n o n o x y g e n sites including t h e 0 6p o s i t i o n o f guanine w h i c h is t h o u g h t t o be significant in m u t a g e n e s i s b y d i r e c t mispairing. T h e m u t a g e n i c i t y o f DMS is b e t t e r explained in t e r m s o f indirect, r e p a i r - d e p e n d e n t processes. With r e s p e c t t o b o t h a l k y l a t i n g activity and genetic effects, striking similarities are f o u n d b e t w e e n DMS and MMS a n d b e t w e e n D E S a n d EMS. In m o s t s y s t e m s w h e r e t h e y have b e e n tested, b o t h DMS a n d D E S are m u t a genic. Results o f m a n y o f t h e m u t a g e n e s i s studies involving these c o m p o u n d s a n d o t h e r a l k y l a t i n g sulfuric acid esters are s u m m a r i z e d in Tables 6, 7, 8, 9 and 10 o f this review. Most d a t a are c o n s i s t e n t w i t h these agents acting p r i m a r i l y as base-pair s u b s t i t u t i o n m u t a g e n s . In t h e case o f D E S , s t r o n g specificity f o r G • C t o A • T t r a n s i t i o n s has b e e n r e p o r t e d in s o m e s y s t e m s b u t has n o t b e e n clearly s u p p o r t e d in s o m e others. L o w levels o f f r a m e s h i f t m u t a t i o n s o f t h e d e l e t i o n t y p e are also likely. In a d d i t i o n t o t h e i n d u c t i o n o f m u t a t i o n s , r e c o m b i n o g e n i c a n d clastogenic effects have b e e n described.

Introduction T h e dialkyl sulfates were a m o n g t h e first chemicals s h o w n t o be m u t a g e n i cally active. T h e initial r e p o r t o f t h e m u t a g e n i c i t y o f these c o m p o u n d s was

65 made in 1947 by I.A. Rapoport who used the sex-linked recessive lethal test in Drosophila [23,246]. Since the pioneering studies of Rapoport, there have been hundreds of genetic studies employing the monofunctional alkylating agents dimethylsulfate (DMS) and diethylsulfate (DES). The files of the Environmental Mutagen Information Center include more than 250 literature citations on DMS and over 400 on DES. In addition to their use in experimental situations, DMS and DES have been used extensively in industry as alkylating agents. The purpose of this review is to provide a comprehensive treatment of the genetic effects of the dialkyl sulfates. An attempt has been made to concentrate on those papers which provide insight into the mechanisms of action o f DMS and DES and those which demonstrate the spectrum of genetic alterations which these compounds induce in a diversity of biological systems. Chemical properties The chemical uses as well as the biological activity of dialkyl sulfates are attributable in large part to their activity as monofunctional alkylating agents. Among the dialkyl sulfates, the dimethyl and diethyl compounds are overwhelmingly the most heavily represented in the chemical literature [313]. In the genetic literature, moreover, the higher dialkyl sulfates are hardly represented. The chemical structures of DMS and DES are shown in Table 1. Names and structures for other alkylating sulfuric acid esters mentioned in this review are also shown in the table. DMS is a colorless, oily liquid which vaporizes readily at 50°C [50]. The vapor is reported to be odorless or to have a slight onion-like odor [50]. In water, one of the methyl groups of DMS is readily hydrolyzed, and the compound is unstable in moist air [50]. The chemical and physical properties of DMS are listed in Table 2. DMS has found extensive use as a methylating agent both in the research laboratory and in industry [79,313]. In industry, it has been used in the manufacture of dyes, perfumes, and a diversity of other products [50]. It has also been used as a solvent in the separation of mineral oils and was once used as a war gas [50]. In addition to serving as alkylating agents, DMS and other dialkyl sulfates have been employed as sulfating and sulfonating reagents [97]. DES, like DMS, is a colorless, oily liquid at room temperature. It tends to darken with age and is reported to have a peppermint odor [198]. The chemical and physical properties of DES are listed in Table 2. The industrial and experimental use of DES is largely as an ethylating agent, and it has been employed in the preparation of a variety o f organic compounds [79]. Literature citations pertaining to some of the industrial uses of DES, as well as DMS, may be found in the books by Fishbein, Flamm and Falk [82]; Browning [50] ; and Fieser and Fieser [79]. Methods that have been employed in the chemical preparation of dialkyl sulfates have also been reviewed [97,198, 313]. A chemical property of sulfuric acid esters that warrants special note for biological studies is their instability in water. With DES, for example, one o f the ethyl groups is readily hydrolyzed [81,118,125,313]. At temperatures

66 TABLE 1 NAMES AND STRUCTURES OF SULFURIC ACID ESTERS Compound

Chemical

Synonyms

Abstracts

Molecular

Structural formula

formula

(CAS)

Number D i m e t h y l sulfate

77-78-1

(DMS)

Sulfuric acid,

C2H604S

CH3--O~ //~O /S CH 3--O ~ O

C4H 1004S

C H 3--CH 2--O\ /~/O \S//

d i m e t h y l ester Methyl sulfate

D i e t h y l sulfate

64-67-5

(DES)

D i p r o p y l sulfate

diethyl ester Ethyl sulfate

CH 3--CH2--O/ - % o

598-05-0

Sulfuric acid, dipropyl ester Propyl sulfate

C6H 1404 S

CH3--(CH2)2--O ~ f O S CH3--(CH2)2--O/ ~ O

625-22-9

Sulfuric acid,

CsH 1804 S

CH3--(CH2)3--O ~ /~O S/~ CH3--(CH2)3--O/ ~ O CH2--O kO ] \S// ~CH2--O / ~ O

(DPS)

D i b u t y l sulfate

Sulfuric acid,

(DBS)

d i b u t y l ester

Butyl sulfate 1 , 2 - E t h y l e n e sulfate

1072-53-3

Ethylene

glycol, cyclic sulfate Glycol sulfate

C2H404S

1073-05-8

1,3-Propanediol, cyclic sulfate

C3H604S

(ESF) 1,3-Propylene sulfate (PSF)

T r i m e t h y l e n e sulfate

1,3-Butylene sulfate (BSF)

4426-50-0

1,3-Butanediol, cyclic sulfate

.CH2 ~ /

O

CH 2

~S///

\

C4H804S

-O

CH 3 [

/CH----<)

C.H2

\// O .~._

~CH2--O/ %O

increasing in increments of 5 ° from 0°C to 40 ° C, the half-lives of DES in water were reported as follows: 59.2, 27.6, 13.1, 6.53, 3.34, 1.80, 1.00, 0.555 and 0.323 h [ 1 1 8 , 1 4 3 ] . In the acid region, the hydrolysis of DES has been found to be independent of pH [89]. The hydrolysis of DMS is even faster than that of DES [ 8 1 , 1 2 5 , 3 1 3 ] , with reports ranging from 1.5 [81] to 5 [313] times faster in water alone, and with larger differences in a variety of other solutions [ 3 1 3 ] . The cyclic sulfuric acid esters, 1,2-ethylene sulfate (ESF), 1,3-propylene sulfate (PSF), and 1,3-butylene sulfate (BSF), are also subject to hydrolysis. The order of these compounds, along with DMS and DES, with respect to instability in water is ESF > DMS > DES > BSF > PSF [37,81]. The order of activity of the same 5 c o m p o u n d s as alkylating agents with 4-(4-nitrobenzyl)-pyridine is ESF > DMS > > DES = PSF :> BSF [81]. The difference in reaction rate between DMS and DES is approx. 70-fold at 30°C [81].

67

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%

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< 0 00

o" O

<

o"

cq

z <

0 e~ 0

68 Toxicology

Acute toxicity Published LDs0 values for DMS and DES, along with those for other sulfuric acid esters, are shown in Table 3. For a general treatment of the toxicology of DMS, the reader is referred to Browning [50]. DMS is particularly hazardous because it lacks warning signals such as a distinct odor or immediate irritation. Exposure to liquid DMS, however, causes blistering and necrosis of the skin. Absorption through the skin is also appreciable and causes systemic poisoning. DMS vapors cause a delayed inflammation of the eyes, mouth, and respiratory tract. Systemic poisoning results in convulsions; paralysis; kidney, liver, and heart damage; and sometimes death. Lethal exposures can occur without being noticed at the time of exposure [50,198]. With the possible exception of oral exposures in rats, for which there is an inconsistency in the scientific literature for DES, the LDs0 values in Table 3 show DES to be less toxic than DMS. In the study of Druckrey et al. [67] in which an LDs0 of 350 mg/kg was reported for DES administered to rats by subcutaneous injection, the deaths occurred 10--20 h following treatment and were due mainly to lung damage. Intestinal damage was, however, also recorded. DBS was found to be of very low toxicity relative to DMS and DES [67]. Carcinogenicity The carcinogenicity of DMS in rats has been studied by Druckrey and his coworkers [67,68]. When DMS was administered in oil by weekly subcutaneous injections of 16 mg/kg (an average total dose of 784 mg/kg), 4 of 6 rats developed tumors at the injection site [68]. Local sarcomas were also found in 7 of 11 rats given weekly subcutaneous doses of 8 mg/kg (an average total dose of 466 mg/kg). Mean survival times were approximately 330 and 500 days for the 16 mg/kg and 8 mg/kg treatment regimens, respectively. Some metastases TABLE 3 LDs0 VALUES FOR SULFURIC ACID ESTERS Compound

Species

Route of administration

LDs0 (mg/kg)

DMS DM S DMS DMS

Rat Rat Rat Mouse

Oral Subcutaneous Intravenous Intraperitoneal

440 100 90 61

DES DES DES DES

Rat Rat Rat Mouse

Oral Subcutaneous Intravenous Intraperitoneal

350a/880 b 350 340 220

DBS ESF PSF BSF

Rat Mouse Mouse Mouse

Subcutaneous Intraperito neal Intraperitoneal Intraperitoneal

5000 26 35 190

Ref.

198 67 67 81 67a/289 b 67 66 81 67 81 81 81

69 to the lungs and a carcinoma of the liver were also reported [68]. The induction of local sarcomas with metastasis to the lungs was confirmed in a later study in which DMS was administered in a single dose of 50 mg/kg by subcutaneous injection [67]. DMS has also been shown to be carcinogenic in rats by respiratory exposure [67]. Treatment involved putting rats into inhalation chambers for 1 h 5 times weekly for 130 days. Squamous carcinomas of the nasal cavity or neurogenic tumors were observed in 5 of 15 surviving rats exposed to 10 PPM DMS and in 3 of 12 exposed to 3 PPM DMS. In contrast to these results, Terracini and Magee, as cited by Swann and Magee [318], found DMS to be only weakly carcinogenic when administered by routes other than repeated subcutaneous injection. Additionally, no tumors were found in rats exposed to DMS b y intravenous injection [67,318]. The negative results may be due to the very short half-life of DMS in water. Consistent with this explanation, Swann [317] demonstrated that no DMS could be detected in the blood of rats 3 min after intravenous injection at a dose of 75 mg/kg. Another negative result for carcinogenicity with DMS is that of Van Duuren at al. [328] who found no papillomas or carcinomas in 20 mice given skin exposures of 0.1 mg DMS 3 times weekly for 475 days. The median survival time was 437 days. Druckrey at al. [68] have described a possible case of human carcinogenesis by DMS. They cite the death of a chemical worker, who was n o t a heavy smoker, from a bronchiogenic carcinoma at 47 years of age, 11 years after an industrial exposure to DMS vapors. Of 6--10 other workers in the same company, 3, and possibly 4, also died of bronchial cancer. Like DMS, DES was found to be carcinogenic in rats following subcutaneous injection [67]. All of 11 animals given weekly injections at a dose of 50 mg/kg (total dose of 1.6 g/kg) developed local sarcomas. When animals were given a weekly dose of 25 mg/kg, 6 of 12 developed local tumors. As in the case of DMS, some metastasis to the lungs occurred. DES given orally to rats by gavage was relatively ineffective for t u m o r induction [67]. After repeated treatments at individual doses of 25 mg/kg and 50 mg/kg (total doses of 1.9 and 3.7 g/kg), only one cancer was observed in 12 animals under each dosage regimen. Both cancers were epithelial carcinomas of the forestomach. Rapid destruction of DES is the probable explanation for its relative ineffectiveness as a carcinogen by oral exposure. In comparative terms, DES is generally regarded as a more p o t e n t carcinogen than DMS [37,67,152,155]. It has been suggested that the weaker carcinogenicity of DMS may be related to its low activity for alkylating oxygen sites in nucleic acids [152]. The lower carcinogenicity generally reported for the methylating agents DMS and methyl methanesulfonate (MMS) relative to that of the methylating nitrosamines and nitrosamides [318] may be explainable in similar terms. The alkylation products formed in nucleic acids by DMS and DES are described later in this review. The production of local sarcomas in rats has been reported following subcutaneous injections of DBS [67]. Although the study was a small one, it appears that DBS is less effective than DMS or DES. The cyclic sulfuric acid ester ESF has also been reported to be carcinogenic. Subcutaneous injections of 0.5 mg

70 ESF given weekly for up to 413 days yielded 22 tumors in 30 mice. Weekly intraperitoneal injections of 0.05 mg produced 6 tumors in 30 mice [328]. Negative results were obtained for ESF in a skin carcinogenesis test in which 0.1 mg of the c o m p o u n d was applied 3 times weekly in an experiment of 456 days duration.

Transplacen tal effects Dialkyl sulfates have been shown to be carcinogenic to fetal rats in transplacental exposures [66,67]. When pregnant rats in the 15th day of gestation were given a single intravenous injection with DMS for a dose of only 20 mg/ kg, 6 [66] or 7 [67] of 59 offspring developed malignant tumors, predominantly of the nervous system. In a comparable study with DES at a dose of 85 mg/kg, 2 [67] or 3 [66] of 30 offspring developed neurogenic tumors. The spontaneous occurrence of this t y p e of t u m o r is reported to be very rare in rats [66,67]. Druckrey et al. [67] report that the fetal nervous system is approximately 50 times more sensitive to carcinogenesis by alkylating agents than tissues of the adult rat. In these studies of transplacental treatment with DMS or DES, no malformations were observed in the newborn [67]. The files of the Environmental Teratology Information Center, Oak Ridge, TN, reveal that the dialkyl sulfates have received little systematic study for transplacental effects other than the causation of neurogenic malignancies in fetal rats. Interaction with DNA

The mutagenicity of DMS and DES is attributable to the fact that they react with DNA as alkylating agents. In this treatment of the alkylating activity of these compounds, I shall restrict m y comments to DNA interactions of likely mutagenic significance rather than considering effects on the totality of DNA chemistry and metabolism. When isolated DNA, phage, or cells are treated with alkylating agents, the major reaction products found in DNA are 7-alkylguanine residues [24,153, 155,165,175,239]. Consistent with this generalization, 7-ethylguanine has been identified as the predominant product in DNA treated with DES [49]. Similarly, 7-methylguanine is the major alkylation product identified from isolated DNA treated with DMS [249]. Alkylation of DNA by dialkyl sulfates has been demonstrated n o t only in vitro b u t also in whole mammals. When Swann and Magee [318] administered DMS (80 mg/kg) to rats b y intravenous injection, 7-methylguanine could be detected in the DNA of several organs. More recently, it has been shown that 7-methylguanine, as well as smaller quantities of 1-methyladenine and 3-methyladenine can be detected in mice given respiratory exposures to DMS [172]. Besides the N-7 position of guanine, sites in DNA bases that have been shown to be susceptible to alkylation include the N-3 and 06 of guanine; the N-l, N-3, and N-7 of adenine; the N-3 and O 4 of thymine; and the N-3 of cytosine [24,155,161,165,274]. Other sites which may be susceptible to alkylation under some conditions are the N-l, N 2, and C-8 of guanine; the N 6 and C-8 of adenine; and the N-4 of cytosine [65]. Recently, the 02 position of

71 TABLE 4 SWAIN--SCOTT s-VALUE OF SELECTED ALKYLATING

AGENTS

Chemical

s

Reaction type

Ref.

Methyl bromide MMS DMS EMS DES MNU iPMS ENU

1.00 a 0.88 0.86 0.67 0.65 b 0.42 0.28 0.26

SN 2 SN2 SN2 SN1/SN 2 SN1/SN 2 SN1 SN1 SN1

256 73 73 156 227 156 256 156

a By definition. b Preliminary determination.

thymine has also been shown to be alkylated by some agents [275]. O:-Alkylcytosine has been reported from single-stranded nucleic acids but not from double-stranded DNA [275]. Since there are single-stranded nucleic acid regions in DNA during replication, however, even products such as O2-alkylcytosine cannot be fully disregarded with respect to possible genetic consequences [275]. In addition to alkylation of nucleic acids, the alkylation of free nucleosides and synthetic polynucleotides has received considerable investigation, and the subject has been reviewed by Singer [274]. DMS and DES have been used extensively in such studies [163,179,274,277,285,310], and several generalizations may be made regarding their behavior. Dialkyl sulfates usually alkylate purine and pyrimidine nucleosides in the following order of reactivity: G > A ~ C ~ > U,T [274]. The most reactive sites in each of the bases are the N-7 of guanine, N-1 of adenine, N-3 of cytosine, N-3 of uracil, and N-3 of thymine [274]. Despite generalizations regarding reactive sites in the purine8 and pyrimidines, there are pronounced differences in the proportions of different alkylation products generated by different alkylating agents. For example, Lawley [155,156] has reported an inverse correlation between the Swain--Scott substrate constant, s [314], of an alkylating agent and the alkylation at oxygen sites in DNA, especially the O6-position of guanine. The substrate constant describes the dependence of alkylation on the nucleophilic strength of the site being attacked [226]. Substrate constants for some of the alkylating agents mentioned in this review are given in Table 4. Agents such as MMS with a relatively high s-value react only through a bimolecular substitution (SN2) type reaction [207]. The kinetics are bimolecular because the SN2 alkylating agent forms a transition complex with the nucleophilic site being alkylated [153]. Agents of low s-value, such as MNU, on the other hand, generate a carbonium ion which then alkylates the nucleophile. Since the rate-limiting step is generation of the carbonium ion, the kinetics are unimolecular (SN1) [153]. Agents of intermediate s-value such as EMS may enter into both unimolecular (SN1) and bimolecular (SN2) substitution reac-

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• '0

,~,.o

73 tions and may therefore be regarded as borderline SN1/SN2 agents. A number of studies have established that alkylating agents which are restricted to the SN2 mechanism are extremely ineffective for producing 06alkylguanine in comparison to agents which are capable of the SN1 reaction [155,156,161,165,175]. Since they are selective for highly nucleophilic sites, such as ring nitrogen atoms [160], SN2 agents are also ineffective for alkylating other oxygen sites in nucleic acids [275]. With SN1 agents such as ethylnitrosourea (ENU), over 80% of total alkylations may be on oxygen sites, especially phosphate groups [275]. For agents of the SN2 type, such as DMS, however, most alkylations are at nitrogen sites [ 155,156,275 ], and alkylation at oxygen sites is very infrequent. Because of the abundance of 7-alkylguanine residues produced in the reaction of alkylating agents with DNA, mispairing attributable to this base was once thought to be the major mechanism of mutation by alkylating agents [24]. Several lines of evidence have suggested, however, that 7-alkylguanine may not be of central importance in the mechanism of mutagenesis and carcinogenesis by alkylating agents [24,156,203]. For example, guanine alkylated at the 7-position is not one of the alkylated bases most likely to be involved in mispairing [155,177]. In addition, both strongly mutagenic alkylating agents such as EMS or MNU and generally weaker mutagens such as DMS and MMS are effective in alkylating the 7-position of guanine. It has been suggested [175], therefore, that 7-alkylguanine may not be the primary pre-mutagenic alkylation product. Specifically, Loveless [175] has suggested that mutagenicity correlates with the formation of O6-alkylguanine rather than N7-alkylguanine. Treating deoxyguanosine with alkylating agents, he found evidence for O6-alkylation by MNU and EMS but not by DMS and MMS [175]. More recently, the formation of O6-alkylguanine was also demonstrated in alkylated DNA, and the results pertaining to differences among alkylating agents were consistent with those of Loveless for deoxyguanosine [155,156,162,163,165]. Subsequent work has provided additional support for the involvement of O 6alkylation in the mutational process [24,92,93,156,239]. Strong correlations have been reported between mutagenicity and O6-alkylguanine formation [65, 155,156,239], and this base is now thought to be of primary importance in mutagenesis by direct mispairing [65,156,160]. Gerchman and Ludlum [92, 93] presented direct evidence that there is extensive misincorporation of bases by RNA polymerase when polyribonucleotide templates contain O6-methylguanine. In a similar study, 7-methylguanine was found to have base-pairing properties very similar to those of guanine [93,177]. The misincorporation attributable to O6-methylguanine involves UMP and to a lesser extent AMP in place of CMP [93]. Thymine alkylated at the O4-position, although apparently an infrequently occurring product reported for only a few mutagens such as MNU [274], is also a likely candidate for mispairing [65,156]. Possible mispairing attributable to 3-alkyladenine [156], 3-alkylguanine [155,156], 3-alkylcytosine [178--181,281], and N%alkylcytosine [65] have also been suggested. In the case of 3-alkylcytosine, misincorporation of UMP and AMP in the RNA polymerase reaction has been reported with a polydeoxyribonucleotide template [179] as well as with a polyribonucleotide template [178,181]. The lack of a simple direct mispairing scheme applicable to 3-alkylcytosine

74 has been discussed by Drake and Baltz [65]. DMS is a very effective alkylating agent. On an equal molarity basis, DMS produces twice as much 7-methylguanine in isolated Bacillus subtilis DNA as does the highly reactive MMS [343]. In terms of total alkylation, moreover, both methylating agents are much more reactive than the corresponding ethylating agents, DES and EMS [49,250,251,274,343]. Ascribing mutagenicity to alkylation products other than 7-alkylguanine helps explain the lack of correlation often observed between mutagenic p o t e n c y and total alkylating activity. DMS and MMS, for example, are more p o t e n t alkylators b u t are often found to be less mutagenic than their ethylating counterparts. Some of the earlier papers on the genetic effects of alkylating agents hypothesized a uniqueness of ethylations to explain the strong genetic activity of particular agents such as DES and EMS [5,174]. Many of the effects once regarded as consequences of the nature of the alkyl group have more recently come to be explained in terms of the mechanism of alkylation and specifically whether or n o t the agent is capable of alkylation by the SN1 mechanism. As its Swain--Scott s-value would indicate (Table 4), DMS may be classified as an alkylating agent that acts almost exclusively b y the SN2 mechanism [162]. Accordingly, it is very ineffective in alkylating oxygen sites [155,156, 162,163] and is similar to MMS in terms of the distribution of methylation products in DNA [61,166]. For b o t h agents the ratio of O~-methylguanine to N7-methylguanine is a b o u t 0.004 [166]; whereas for the methylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), the O6/N7 ratio is 0.1 [156]. As will become evident, DMS is also similar to MMS in its genetic effects. The behavior of DES suggests that this c o m p o u n d should be classified with agents like EMS which are capable of SN1 reaction [162,239]. Kugmierek and Singer [152] found that DES was much more effective than DMS in alkylating oxygen sites in polyuridylic acid and produced a spectrum of alkylation products very similar to that produced by EMS. A preliminary determination of the s-value for DES [227,326] further supports the interpretation that DES is quite like EMS in reactivity and unlike DMS and MMS. The substrate constant for DES, as shown in Table 4, suggests that this c o m p o u n d be classified as an intermediate SN1/SN2 alkylating agent. Table 5, adapted from Singer [ 274], is a compilation of data on the distribution of alkylation products among the bases of R N A and DNA following treatment with DMS or DES. N o t all rows add up to 100% because in some cases there are unidentified products, or the remainders represent phosphate alkylation. Data for MNU and ENU, which are typical SN1 agents, are included for purposes of comparison. The data show that although DES does n o t alkylate the O6-position of guanine nearly as effectively as typical SN1 agents do, it is certainly more effective in this regard than is DMS [274,275,283]. For example, in R N A from tobacco mosaic virus (TMV), 2% of total ethylation by DES was in the form of O6-eth ylguanine whereas no O~-methylguanine could be detected following alkylation with DMS [283]. The spectrum and relative frequency of alkylation products formed by DES is very similar to that formed by EMS [283]. ' Due to the differences in alkylation products in DNA, SN2 reagents such as DMS or MMS may be expected to be less mutagenic than agents such as DES or

75 EMS which are capable of SN1 reactions. A considerable body of literature can be found in support of this hypothesis [33,174,239,250], and SN1 agents are often regarded as being more mutagenic or carcinogenic than related SN2 compounds [306]. In fact, it has been suggested that there is a general inverse correlation between mutagenic efficiency and s-value [160,219,226,329]. Generalizations regarding the relative mutagenicity of alkylating agents must, however, be drawn cautiously because of differences among agents with respect to overall toxicity, predominant mechanisms of toxicity, kinetics of inactivation, kinetics of mutation induction, distribution of the agent in cells and tissues, and reactivity of the agent under different conditions [226,326]. There are, in addition, important differences between agents with regard to the effects of repair systems on the lesions induced [65]. A number of exceptions to the generalization that mutagenic, as well as carcinogenic [160], potency correlates with SN1 reactivity have been published. Isopropyl methanesulfonate, an agent of low s-value, is reported to be a less efficient mutagen than EMS [54,226]. Rhaese and Boetker [250] reported that MMS is 5--10 times more effective than EMS for inducing mutations in transforming DNA of Bacillus subtilis. In E. coli WP2, Strauss [304] found MMS and EMS approximately equally effective for inducing tryptophan reversions. Comparisons of the relative mutagenicity of DMS versus DES or of MMS versus EMS are not completely straightforward, however, because of the markedly different toxicities observed between the two members of each pair of compounds [5,343] with the methylating agents exhibiting greater toxicity. Zamenhof and Arikawa [343] reported, for example, that at equal levels of survival, EMS was more mutagenic than MMS in Bacillus subtilis. When the comparison is made on the basis of equal chemical concentration, however, MMS is the more mutagenic of the two compounds [343]. Even these results are not easily generalized because the toxicity of an alkylating agent may depend at least in part upon the anionic residue. Zamenhof and Arikawa [343] report, for example, that the order of effectiveness for killing vegative cells of Bacillus subtilis is sulfate > methanesulfonate > iodide for the methylating agents, but sulfate > iodide > methanesulfonate for the ethylating agents. Thus, the result for mutagenicity of MMS and EMS at equal survival levels cannot be confidently extrapolated to DMS and DES. In fact, the data of Zamenhof and Arikawa [343] indicate that, unlike the methanesulfonates, DMS is more mutagenic than DES for Bacillus subtilis at equal levels of survival. Because they produce little alkylation at oxygen sites, the mutagenicity of SN2 reactants like DMS must be explained differently from that of agents capable of the SN1 reaction. One suggestion has been that the mutagenicity of SN2 agents may involve mispairing of the relatively small amounts of 3-methylguanine formed in DNA [155,161]. Other factors, however, must also be considered, not the least of which is the role of DNA repair processes in mutagenesis. In fact, it now appears that there are two major classes of mechanisms of mutagenicity of alkylating agents [65]. The first is mutagenesis by direct mispairing, and is attributable to O6-alkylguanine and other alkylated bases which have altered pairing specificity. The second class is indirect mutagenesis in which the occurrence of mutation depends upon errors in enzymatic repair

76

processes. Indirect mutagenesis is thought to contribute substantially to the genetic activity of SN2 agents [65]. Although no longer thought to be of primary importance in mutagenesis by direct mispairing, the abundant N7-alkylguanine residues formed in alkylated DNA should not be regarded as completely without consequence. Alkylation of the N7-position of guanine apparently weakens the glycosidic linkage and can lead to loss of the purine from DNA [24,82,153,156,157]. Depurination has also been associated with other N-7 and N-3 alkylation products [156] such as 3-alkyladenine [157,274]. In addition to spontaneous depurination, removal of alkylated bases from DNA in vivo apparently occurs by enzymatic repair processes [156]. Weakening of glycosidic linkages has also been reported for O2-alkylthymine [275]. Although depyrimidination due to this base is likely, its genetic consequences are uncertain. Even for depurination, which has frequently been proposed as a cause of transversions and other mutations induced by alkylating agents [33,82,86], the genetic significance has not been clearly established [24]. Several arguments against a role for depurination in mutagenicity have been outlined in earlier reviews [64,65]. While a major role for depurination in mutagenesis seems unlikely, its contribution to lethality due to resultant strand breakage [85,87,156--158,250, 308,327], possibly including double-strand breakage [327], is more likely. In fact, the greater toxicity of some methylating agents relative to the equivalent ethylating agents [24,157,343], or of SN2 reagents relative to SN1 reagents [156], may be attributable at least in part to their greater efficiency for alkylating the nitrogen atoms of purines and the resultant depurination. There is probably, in addition, a lower ratio of DNA alkylation to protein alkylation with agents of high s-value [226], and protein alkylation may well contribute to lethality. The greater cytotoxicity of DMS relative to DES is consistent with such explanations. It has been suggested that besides its involvement in lethal effects, depurination may contribute to chromosome breakage [24,85,87] and to occasional base-pair deletions induced by alkylating agents [224]. Apart from causing depurination, some alkylations per se may contribute to lethality [156]. Singer et al. [286] have suggested that 7-alkylguanine produced in the RNA of TMV by DMS or DES may be inactivating without strand breakage. Alkylations which block normal base-pair hydrogen bonding have been reported to have lethal consequences for bacteriophage R17 RNA treated with DMS [156,272]. Specifically, methylation of the Nl-position of adenine and the N3-position of cytosine was shown to be inactivating [272]. According to Lawley [156], it is therefore likely that alkylations of the Nl-position of adenine, the N-3 of cytosine, or the N-3 of thymine, while not extensive, could contribute to inactivation of DNA. Many alkylating agents react not only with the bases of DNA but also with the phosphate groups to form phosphotriesters [31,154,155,273,275]. Because of their greater reactivity for oxygen sites in DNA, agents of low s-value are relatively efficient in phosphate alkylation compared to agents of high s-value [156]. Bannon and Verley [31] reported that alkylation of phosphates represents 15% of total DNA alkylation by EMS and only 1% by MMS. With SN1 reagents like ENU, phosphotriester formation may represent two-thirds of total

77 alkylation [274,283]. Consistent with this generalization regarding SN1 and SN2 agents, DMS has been reported not to cause phosphotriester formation in DNA [273]. DMS has also been shown to be inactive in a test for phosphate alkylation based upon strand breakage in phage RNA [271]. For DES, on the other hand, phosphate ethylation has been found to constitute 16--20% of total alkylation of HeLa cell DNA [311]. Consistent with this result, DES has been found to be more effective than DMS for alkylation of phosphate groups in polyuridylic acid [152]. DES was roughly equivalent to EMS in this regard [152]. The data for alkylation of phosphate groups in nucleic acids by DES are, however, still incomplete. For example, Singer and Fraenkel-Conrat [283] found that DES yielded a high level of phosphate alkylation in HeLa cell RNA where many terminal phosphate groups are exposed but not in the RNA of TMV. In general, however, one would expect DES, like EMS, to react with the phosphates of nucleic acids to a moderate extent while phosphate alkylation by DMS is negligible. A major mutagenic role for phosphotriesters is generally regarded as unlikely [64,250,274]. DNA strand breakage which may result from hydrolysis of alkylated phosphate groups [85,251,274] may, however, contribute to lethality [250,274,275] and perhaps to chromosome breakage as well [87]. Effects of phosphotriester formation through changes in nucleic acid conformation or altered interaction with complementary polynucleotides or with enzymes have also been proposed [311]. In this regard, evidence has been presented for phosphate alkylation, per se, causing inactivation of RNA of TMV without causing strand breakage [286].

Repair processes Repair o f alkylated DNA It has been known for some time that alkylated bases are readily removed from DNA. Moreover, the rates of removal of such bases in vivo are higher than can" be explained solely by depurination as occurs in vitro [156,191,274]. Apparently, alkylated bases are efficiently removed from cellular DNA by enzymatic repair processes. There are distinct differences in the efficiency of removal of different alkylation products in DNA [306]. In E. coli, for example, it has been found that excision of methylated bases occurs following treatment with MNNG [159]. This excision process, moreover, removes O6-methylguanine residues but not 7-methylguanine residues. Among methylated bases removed following treatment of E. coli with MNNG, the order of efficiency of excision was 3-methyladenine > O6-methylguanine > methylated pyrimidines ~> 7-methylguanine [159]. Consistent with these results, Lawley and Warren [166] have found that, after DMS treatment, E. coli repair processes effectively remove 3-methyladenine and 3-methylguanine residues but not purines methylated in the 7-position. Endonuclease II from E. coil has been shown to release O6-methylguanine and 3-methyladenine but not 7-methylguanine from DNA alkylated in vitro with MNU [136]. For DNA methylated by DMS, endonuclease II releases

78 3-methyladenine but not 7-methylguanine [137]. Rapid loss of 3-methyladenine relative to 7-methylguanine or O6-methylguanine has also been reported for rat-liver DNA methylated in vivo with dimethylnitrosamine [60]. It has been suggested [166] that N3-purine methylations may block DNA-template activity and stimulate enzymatic repair processes. There are reports in the literature that methylated bases formed by MMS treatment of E. coli are not subject to excision repair [141] or that the E. coli and Bacillus subtilis repair systems do not recognize methyl groups following treatment with MMS [240]. These reports are consistent with the inability of the excision system to remove the abundant 7-alkylguanine residues and the lack of O6-alkylation caused by MMS. Low levels of N3-alkylated purine removal may have gone undetected. That some of the lesions induced by SN2 methylating agents interact with repair systems is suggested by the fact that a p o l A strain of E. coli is more sensitive to killing by MMS [83,289] and DMS [83] than is the wild-type. There is no reason to conclude, therefore, that there is a fundamental difference in DNA-interactions or repair-response between DMS and MMS. It seems reasonable to expect, based upon the differences between SN1 and SN2 agents, that DMS and MMS would be quite similar in terms of reparability of lesions and that DES would be more like MNNG in that the effects of O6-alkylation would be recognized. In addition to the enzymatic removal of 3-methylpurines from DNA treated with DMS, several other lines of evidence suggest the action of repair systems on damage induced by dialkyl sulfates. For example, DNA-repair synthesis has been reported in Chinese hamster cells treated with DMS [61]. In Drosophila, strains which are resistant to mutagenic effects of ionizing radiation are reported to be resistant to the induction of lethals by DES as well. Common repair mechanisms provide one possible explanation [193]. That bacterial DNA-repair deficiencies confer increased sensitivity to killing [83] or to inactivation of transforming DNA [41] by DMS suggests the repairability of induced lesions. The association of repair deficiencies with enhanced induction of sister-chromatid exchange in human cells by DMS [338] may be similarly interpreted. Effects of a respiratory deficiency on liquid-holding recovery and recombinogenicity in DES-treated yeasts provide additional indirect evidence of repair [346]. More information on these studies is included in the appropriate section for each test organism. Error-prone repair

Repair functions have important implications for mutagenicity. A recent review by E. Witkin [337] clearly outlines the central role of an inducible repair system (S.O.S. repair} dependent upon recA ÷ and l e x A ÷ gene function in the mutagenicity of ultraviolet light. Comparable r e c A * - d e p e n d e n c e has been found for the induction of mutation by certain chemicals. The mutagenicity of MM$ depends upon intact recA ÷ function in E. coli whereas that of EMS is largely independent of recA ÷ [141]. In phage T4, the mutagenicity of MMS may similarly be explained largely in terms of errorprone repair [65,104]. In E. coil the mutagenicity of MNNG depends only partly upon recA ÷ gene function, suggesting a combination of the recA÷-dependent and r e c A ÷ - i n d e p e n d e n t mechanisms of action [141]. It has been pointed

79 o u t that the mutagenicity of typical SN2 alkylating agents such as DMS generally proceeds b y a mechanism involving error-prone repair whereas agents capable of SN1 alkylation can induce mutations by direct mispairing independently of repair [219]. One might predict, therefore, that loss of recA ÷ gene function would have a major effect on the mutagenicity of DMS and less effect on that of DES. Mutagens which act by the indirect, repair-dependent mechanism do not show the same specificity for transitions as do those that act by indirect mispairing. Rather, they induce a variety of alterations including transitions, transversions, frameshifts, and deletions [65]. The greater specificity of DES than DMS for G • C -~ A • T transitions in Saccharomyces [239] may well be explainable in terms of DES acting by the direct mechanism and DMS by the indirect mechanism. In this regard, Prakash and Sherman [239] have proposed that both the extent of mutagenicity and the specificity for G • C to A • T transitions depends upon the amount of O6-alkylguanine formed in DNA. It is n o t e w o r t h y that genes involved in repair in yeasts have been shown to affect mutagenicity of agents which act by a recA÷-independent mechanism as well as those which are recA÷-dependent for mutagenicity. Specifically, Prakash [238] has shown that incorporation of the rad-6 and rad-9 alleles into the cycl131 tester strain of Saccharomyces cerevisiae causes extreme reduction or elimination of mutability by DMS, DES, EMS, MNNG, and a number of other compounds. The relationship of rad-6 and rad-9 to other known repair deficiencies remains to be determined.

Overview of mutagenicity data DMS and DES are genetically active in a diversity of organisms and test systems. Of the two compounds, DES is frequently regarded as the more effective mutagen and has been used more extensively in genetic studies. Differences in the genetic effects of DMS and DES may be related to a considerable extent to differences in the alkylation products formed by the two c o m p o u n d s in DNA. Tables 6 and 7 present a broad overview of genetic effects reported for DMS and DES respectively. The tables a t t e m p t to provide a quick reference on the availability of data for several major genetic endpoints as well as for organisms throughout the phylogenetic hierarchy. Table 8 provides a more thorough synopsis of studies conducted on the genetic effects of DMS in various organisms and test systems. Citations to key papers are included in the table. Comparable information for DES is presented in Table 9. Table 10 is a synopsis of mutagenicity tests conducted with other alkylating sulfuric acid esters. More details on the studies included in these tables and generalizations that may be drawn from them can be found in the appropriate sections of the text. Studies in which dialkyl sulfates were merely used to obtain mutants but which do n o t provide information on mechanisms of action or dose-response relationships and do n o t represent the only data for the effects of these compounds in a particular type of organism or test system have been excluded from

8O TABLE

6

GENETIC

EFFECTS

REPORTED

FOR

DMS

Type of test

Result

S e l e c t e d ref. a

R N A virus Mutation

+

280

Prokaryotes Mutation Transforming DNA mutation R e p a i r assay ProPhage i n d u c t i o n

+ +

37 38

+ +

83 78

Fungi Mutation

+

239

Vascular plants Mutation Chromosome aberrations

+/-+

73/221 173

Insects Mutation

+

5

Fish Mutation Chromosome aberrations

+ +

323 322

+ +

59 261

+ +

338 56

M a m m a l i a n cells Mutation Chromosome aberrations Sister-chromatid exchanges Repair replication

Whole m a m m a l Dominant lethals Testicular DNA synthesis inhibition

--

77

+

264

a One literature c i t a t i o n is given for e a c h p o s i t i v e or negative result e n t e r e d i n t o the t a b l e . F o r m o r e e x t e n s i v e c i t a t i o n s , r e f e r t o Table 8 .

the tables. Some such studies are mentioned in the text, however, because they contribute to an overview of the extensiveness of use of dialkyl sulfates in genetic studies. Genetic effects in viruses In the early experiments of Loveless [ 1 7 4 ] , DES was shown to be active in inducing r-mutants (rapid lysis) in phage T2 of E. coli. EMS was similarly found to be active, but MMS was not. The genetic activity of DES has also been reported for phage T4 [225] in which EMS is more mutagenic than MMS [65]. Although data for DMS in phage have not been reported, the parallel to EMS and MMS would lead one to expect its activity to be less than that of DES. Unexpectedly, DES was found to be non-mutagenic in tobacco mosaic virus (TMV) whether treatment was performed on isolated R N A or on the intact virus [ 2 7 9 , 2 8 0 ] . The generally similar ethylating agent EMS, however, was found to be active [ 2 7 9 , 2 8 0 ] . Unlike the result with DES, DM8 is mutagenic in TMV [ 8 4 , 2 7 9 , 3 2 5 ] . When DMS-treated viruses or reconstituted viruses containing R N A that had been

81 TABLE 7 GENETIC EFFECTS

REPORTED

FOR DES

T y p e of test

Result

Selected ref. a

D N A virus Mutation

+

174

R N A virus Mutation

--

280

+

196

Prokaryotes Mutation Deletions Repair assay Prophage i n d u c t i o n

+/--

90/13

+/-+

217/15 194

+ + +

239 112 350

C h r o m o s o m e aberrations

+ + +

119 30 243

Insects Mutation Translocations Male crossing-over D o m i n a n t lethals

+ +/-+ +

230 210/228 228 212

M a m m a l i a n cells Mutation Repair r e p l i c a t i o n

+ +

Whole m a m m a l D o m i n a n t lethals Testicular D N A synthesis i n h i b i t i o n Chromosome exchanges (meiotic) C h r o m o s o m a l abnormalities ( m i t o t i c ) Specific-locus m u t a t i o n s

+ + -+/-+

Fungi Mutation C h r o m o s o m a l rearrangements Mitotic r e c o m b i n a t i o n Vascular p l a n t s Mutation Paramutation

59 56

184 264 169 312/312 183

a One literature c i t a t i o n is given for each positive or negative result entered i n t o the table. F o r m o r e e x t e n s i v e citations, refer t o T a b l e 9 .

alkylated in vitro with DMS were applied to Nicotiana sylvestris, mutants of altered pathogenicity could be detected [ 8 4 , 2 7 8 , 2 7 9 ] . Mutants which produced local lesions on inoculated leaves were recovered from a parent strain which produced a systemic disease in the host but not local lesions [84]. Mutagen exposures were conducted so as to cause 60--99% decrease in infectivity [2801. Although active, DMS is a rather low-level mutagen in TMV [ 8 4 , 2 7 9 ] . Following exposure of the intact virus or isolated R N A to DMS, increases in mutation frequency were 3- and 4-fold resp. [ 2 7 9 , 2 8 0 ] . The corresponding increases caused by nitrous acid were l l 0 - f o l d for the intact virus and 34-fold for the isolated nucleic acid [ 2 7 9 , 2 8 0 ] . The methylating agent MNNG, in contrast to DMS, is reported to be highly mutagenic in the intact virus (60-fold increase) but not in isolated R N A (2-fold increase) [ 2 7 6 , 2 7 9 , 2 8 0 , 2 8 4 ] . MMS is reported to be roughly equivalent to DMS in mutagenic activity, showing 4- and 5-fold

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89 increases in intact virus and isolated RNA, resp. [279]. Singer [274] has pointed o u t that the greater mutagenicity of MNNG than DMS or MMS in this system correlates with the formation of 3-methylcytosine, which represents 35% of total R N A methylation with MNNG [274] b u t less than 10% with DMS [283]. Singer and Fraenkel-Conrat [282] have demonstrated heritable amino acid changes in the coat protein of many TMV mutants detected by altered pathogenicity in Nicotiana sylvestris. Among these mutants are mutants induced by DMS. One cannot conclude from the data presented what specific codon changes are induced by DMS because the data for DMS are pooled with those from MMS and MNNG. For the alkylating agents in general, however, a variety of base-pair substitutions, involving all 4 of the R N A bases at the mutant site, were detected [282]. A mutagenic effect of DMS in a less well-characterized viral genetic system was reported by Solyanik et al. [291] for Eastern equine encephalomyelitis virus. Changes in plaque size on cultured chick-embryo cells and in pathogenicity in rodents were used as indices of mutagenic activity. The variant characteristics were shown to be stable in repeated transfers in chick-embryo cell cultures [292]. The induction of mutations b y DMS in an animal virus has also been reported b y Thiry [321] who studied variants of Newcastle disease virus which differ from wild-type in stainability of plaques on chick-embryo cells by the dye, neutral red. Genetic effects in prokaryotes

Induction of base-pair substitutions in bacteria The most widely used of all systems for the detection of mutations is that developed b y Ames and his coworkers based upon reversion in histidine mutants of Salmonella typhimurium [14--16,195,196]. One of the alleles used in the Ames test is the base-pair substitution mutation hisG46. DES has been shown to revert this allele [14,51]. To increase the sensitivity of the test, Ames developed strain T A 1 5 3 0 which contains the hisG46 allele and a deletion through gal, chl, bio and uvrB. The deletion, which confers a partial deficiency in the bacterial lipopolysaccharide coat due to loss of the gal function and a deficiency for excision repair due to uvrB [ 15], causes a 7-fold increase in sensitivity to DES [14]. Strain TA1950, which includes the hisG46 mutation and a deletion through uvrB b u t n o t gal, is also readily revertible b y DES [336]. Quantitative comparisons of this strain with hisG46 were n o t made, however, and the enhnaced mutagenesis of strain TA1530 relative to hisG46 cannot be attributed specifically to loss o f gal function or of uvrB. Strain TA1535, one of the tester strains n o w in c o m m o n use, includes, in addition to the hisG46 allele and the deletion through uvrB, a mutation conferring a deficiency in the bacterial mucopolysaccharide coat. This mutation, rfa, provides for increased permeability to a variety of large molecules. Strain TA100 has the same chromosomal genotype as strain TA1535 b u t includes the pKM101 plasmid. As expected, DES is active in reverting the hisG46 allele in strains TA1535 and TA100 [196]. In studies with strain TA1535, DES is a rather p o t e n t mutagen in the Ames

90 test. When 5000 pg of DES were added per plate, a mutant count of 14 762 induced revertants per plate was reported. In comparison, EMS, at 5000 pg per plate, induced only 220 revertants per plate in the same strain [196]. Reversion in strain T A 1 5 3 5 indicates that DES induces base-pair substitutions. Strain TA100, because of decreased specificity associated with the pKM101 plasmid, is less diagnostic for mechanism of mutations [ 196]. Strain T A 1 0 0 has been incorporated into the standard series of tester strains r e c o m m e n d e d by Ames [16] because it is more sensitive to the action of many chemicals than is its parent strain TA1535. In one study, the mutagenicity of DES has been reported to be greater in strain TA100 than in strain TA1535 [287]. In this study, however, the activity of DES in strain TA1535 is surprisingly weak. Elsewhere, strain TA100 is reported to be no more sensitive than strain TA1535 to mutagenesis by DES; in fact, it is probably less sensitive [196]. Although the mechanism b y which the pKM101 plasmid increases the sensitivity to many mutagens is incompletely understood, it seems to involve a recombination-dependent, error-prone repair process [196]. The observation that sensitivity to DES is n o t increased by the presence of the plasmid is consistent with DES inducing mutations b y a nonrepair-dependent mechanism. Like DES, DMS induces reversions in strain TA1535 of Salmonella typhimurium [37,287]. In the Ames test, strains T A 1 0 0 and TA1535 are of roughly equal sensitivity to mutagenesis by EMS, but strain TA100 has enhanced sensitivity to mutagenesis b y MMS [196]. One would expect that DMS, being an SN2 agent, would be like MMS in terms of the relative sensitivity of TA1535 and TA100. While data for DMS in the Ames test are n o t extensive, one report in the literature shows some enhancement of mutagenic response in T A 1 0 0 relative to that in TA1535 [287]. In addition to DMS and DES, several cyclic sulfuric acid esters have been evaluated for mutagenicity in Salmonella typhimurium. The monofunctional alkylating agents, 1,2-ethylene sulfate (ESF), 1,3-propylene sulfate (PSF), and 1,3-butylene sulfate (BSF) are all active in strain TA1535, indicating induction of base-pair substitutions [37]. In terms of concentrations required to detect mutagenicity and numbers of revertants in plate tests, the order of activity in strain TA1535 was as follows: PSF > BSF > ESF > DES > DMS [37]. While such comparisons are complicated by a number of factors, not the least of which is instability in water, the data clearly show the cyclic sulfates to be effective mutagens in Salmonella. The lower mutagenicity of ESF relative to PSF and BSF may be related to its very high rate of hydrolysis. In E. coli, Alderson [5] has shown that both DMS and DES induce mutations conferring resistance to phage T4. When equal concentrations of the two c o m p o u n d s (10 -3 M) were used, higher mutation frequencies were recovered with DMS. After a 15-min treatment, however, survival was 100% with DES and only 2% with DMS. When the dose of DES was increased to 0.1 M, which results in 20% survival, the mutation frequency was a b o u t 500 phage-resistant mutants per 10 ~ cells. This is more than 5 times the highest frequency reported with DMS [5]. The great difference in toxicity of DMS and DES complicates the comparison of their mutagenicities. On an equimolar basis, DMS induced a higher frequency of mutations. At comparable levels of killing, however, DES was the more effective mutagen.

91 Dose
92

Induction o f frameshift mutations in bacteria In contrast to the results for strain TA1535, DES is reported to be inactive in the frameshift strains TA1537 [287], TA1538 [196,287], and TA98 [196, 287]. The results obtained in these widely used tester strains are consistent in terms of mutational specificity with earlier results [51] for the original strains hisG46 and hisC3076 upon which strains TA1535 and TA1537, respectively, are based. DES was found to revert hisG46 but was inactive on the frameshift allele hisC3076. For DMS, there is an inconsistency in the literature with respect to revertibility of frameshift tester strains. DMS has been reported both as positive [287] and as negative [37] in the Ames' frameshift strains TA1537 and TA1538. The cyclic sulfates ESF, PSF and BSF, like DES, are reported to be active in strain TA1535 but inactive in strains TA1537 and TA1538 [37]. An exception to the notion that DES is specific for base-pair substitutions may be found in the data of Hartman and his coworkers [115,224]. DES, as expected, was f o u n d to revert a variety of missense and nonsense mutations in the histidine operon of Salmonella typhimurium. It can also revert, however, the hisD3052 allele which contains a frameshift m u t a t i o n [ 115]. In the finely designed experiments of Oeschger and Hartman [224], it was found that one class of mutations induced by the frameshift mutagens ICR-191 or ICR-364-OH could be reverted both by frameshift mutagens and by alkylating agents, including DES. The behavior of this class of mutants, which included 45% of the ICR-induced mutations, suggested that these mutants contained base-pair additions. Oeschger and Hartman [224] therefore suggested that the mechanism of reversion in these mutants is base-pair deletion. A carefully reasoned argument has been presented [224] that the reversion of frameshifts by DES or MNNG is not attributable to suppression of or mutation in nonsense codons generated by the alteration in reading frame. As a rough estimate of the proportion of frameshift mutations induced, Oeschger and Hartman [224] calculated that an MNNG treatment causing a 185-fold increase in reversion frequency in a base-pair substitution strain caused a 15fold increase in 4 frameshift strains. Depurination provides a possible mechanism whereby alkylating agents can induce a low level of frameshift mutations of the base-deletion t y p e [24,224]. Alternatively, the mechanism may be based upon misrepair of lesions in DNA [224]. Direct evidence for the induction of frameshift mutations by mutagens which induce predominantly base-pair substitutions is provided by the work of Yourno and Heath [342]. Reversion by MNNG of hisD3018, a frameshift mutation containing one extra nucleotide pair, can restore the correct amino acid sequence in histidine dehydrogenase, coded for by the hisD gene [342]. Although a comparable direct demonstration of base-pair deletions induced by DES has n o t been published, the available evidence is quite convincing that, in addition to inducing base-pair substitutions, a lower level of frameshift mutagenesis by DES does occur. The induction of reversions in frameshift tester strains frequently involves considerable specificity for particular sequences near the m u t a n t site [15,16]. According to Hartman [114], DES is able to revert frameshift mutations which have a run of cytidylic acid residues in the same strand near the m u t a n t site,

93 and also those which have a series of alternating G's and C's near the mutant site. The frequencies of reversion are, of course, lower for frameshift mutations than for base-pair substitutions. Additional reversion data support the same conclusions with respect to the induction of frameshift mutations b y DES. In a study of 152 hisD mutants of Salmonella typhimurium, it was reported [112] that 7 mutants that were classified as frameshifts, including one that is highly revertible by ICR-191, could be reverted by DES. As expected, all the nonsense mutations in these 152 mutants were highly revertible by DES. Similarly, in a study of 212 histidine mutants of E. coli [90], 4 mutants which are revertible by DES, MNNG, and ICR-191 were found. The majority of the mutants which were revertible by DES and MNNG, however, were not subject to reversion b y ICR-191. In a study o f araB mutants of E. coli [ 132 ], most alleles that were revertible by DES were also reverted by EMS but not by ICR-170 or ICR-191. Several lines of evidence indicated that these alleles contained missense or nonsense mutations [132]. One mutant, highly revertible by the ICR compounds and presumably a frameshift, however, was also weakly revertible by DES and EMS. It has also been reported in a meeting abstract [55] that 12 lacZ frameshift mutations of E. coli are revertible by DES. Not all of the studies indicating reversion of frameshift mutations by DES explore alternative interpretations of the data as does the paper of Oeschger and Hartman [224]. Taken in their totality, however, such studies support the conclusion that DES induces a low level of frameshift mutations in addition to its efficient induction of base-pair substitutions. Induction o f deletions in bacteria An interesting result from reversion studies in E. coli [90] is that 11 o u t of 25 his mutations induced by DES were nonrevertible. Genetic evidence has been presented [90] indicating that such mutants contain extended deletions in the histidine region. It might seem, then, that DES is also capable of inducing deletions in bacteria. This conclusion should be qualified, however, by noting a possible peculiarity of the system. Deletions were found to be much more c o m m o n among E. coli his mutants than among Salmonella mutants [90,100], and point mutations in the hisD or hisB genes of E. coli were very u n c o m m o n relative to their occurrence in Salmonella [90]. The system, therefore, may magnify the capacity of an agent to induce deletions. Alper and Ames [13] have described a positive selection for long deletions in Salmonella. By selecting for simultaneous resistance to 2-deoxygalactose and potassium chlorate, one selects for bacteria which contain deletions of the gal--chl region of the Salmonella chromosome. While agents which are known to cause deletions, such as nitrous acid [262], are active in this system, DES is not [13]. Other mu rational studies in bacteria A bacterial forward mutation test in which the dialkyl sulfates have been evaluated for mutagenicity is that based upon resistance to 8-azaguanine in Salmonella typhimurium [287]. Both DMS and D E s were shown to be active in this test, and mutagenic responses were dose dependent. Using azide-resis-

94 tance and reversion of histidine a u x o t r o p h y as indicators of mutagenicity, Z a m e n h o f and Arikawa [343] studied mutagenesis by DMS and DES in Bacillus subtilis. In contrast to results ontained in several other systems, t h e y found DMS to be more potent a mutagen than DES at equal levels of survival [343]. In E. coli, Strauss and Okubo [307] reported dose-dependent increases in reversion frequency in a t r y p t o p h a n auxotroph treated with DES. Even with treatment times up to 75 min, little cell killing was observed, and evidence was provided that cell divisions are required for expression of DES-induced pre-mutational lesions as mutations [307]. In addition to studying genetic effects of DES, Strauss [305] has reported an apparent physiological alteration in cellpermeability caused by DES in E. coli. The mutagenesis data of Strauss and Okubo [307] for DES have been interpreted [253] in terms of delayed mutagenic effects. The possible occurrence of such effects following t r e a t m e n t with alkylating agents is supported by studies indicating delayed mutagenesis in phage T4 [103,149]. Ronen [253] has studied the delayed appearance of revertants for the Ieu524 allele of Salmonella typhimurium following treatment with DES. The experiments were based upon the time required for tubes of selective medium to become turbid [253]. Although the appearance of mutants may continue after exposure to DES has stopped, it is not clear to which part of the mutational process such delay may be ascribed. Both fixation of pre-mutational lesions in DNA and physiological processes involved in the phenotypic expression of the new mutations can contribute to the delayed appearance of mutants. In subsequent experiments on the delayed appearance of revertants in a series of leu mutants of Salmonella, Ronen [254] reported that mutants may be classified into two groups based upon pattern of reversion. In one group, including 1eu524, delayed m u t a t i o n is observed. In the other group, it is not. Ronen's interpretation of the results is that the group which exhibits delay reverts by A • T to G • C substitutions occurring as a consequence of depurination [254]. The group n o t exhibiting delay was proposed to revert by direct mispairing with a G " C base pair at the m u t a n t site [254]. Unfortunately, there is little solid evidence regarding the reversion mechanisms in the leu mutants used in these strains, and other interpretations of the data are possible [64]. Because DMS and DES exert their genetic effects through alkylation of DNA, one might expect t h a t the effectiveness of mutagenesis by such agents would depend upon the extent of exposure of DNA to attack. In a study by Brock [46], it was found t h a t a missense m u t a t i o n in the ~-galactosidase gene (lacZ) of E. coli could be induced to revert by several alkylating agents, including DES, more readily when the lac operon is induced than when it is repressed. A treatment with DES giving a 6-fold increase in m u t a t i o n frequency in the uninduced operon gave a 39-fold increase in the induced operon. A comparable enhancement of mutagenesis by induction of the operon was n o t observed for ~'-rays. The explanation suggested by Brock [46] is that the separation of the DNA strands for transcription also exposes the bases for alkylation. Another possibility, suggested by Lawley [156], is that transcribed DNA may be more susceptible to mutagenesis by DES because enzymatic excision could be blocked during the synthesis of RNA or of DNA.

95 Experimental conditions shown to quantitatively modify the effectiveness of DES in bacterial tests include treatment in hyperbaric environments [336] and growth in the presence of caffeine following DES treatment [130]. In the latter study, caffeine caused a slight reduction in the frequency of streptomycinindependent mutants recovered in a streptomycin-dependent strain of E. coli at all DES doses [130]. Because the dialkyl sulfates are mutagenic without metabolic activation, relatively little work has been done regarding the effects of mammalian activation systems on mutagenesis by these compounds. DMS and DES, at a dose of 2500 pmole/kg, have been shown, however, to revert the hisG46 allele in Salmonella typhimurium strain TA1950 in the host-mediated assay in mice [37]. The bacteria were injected into the mice intraperitoneally and the chemicals were administered orally. Similarly, the cyclic sulfates PSF and BSF were active at doses of 10 and 50 pmole/kg, resp. The highly unstable ESF, on the other hand, was only weakly active even at doses as high as 5000 pmole/kg [37]. Differences among compounds in such studies involve differences in water solubility, instability in water, and pharmacological disposition as well as the inherent mutagenic properties of the chemicals. DES has been widely used to obtain mutants in those bacterial species that are extensively used in genetic studies. For example, in Salmonella typhimurium DES has been employed for the induction of mutations to D-histidine utilization in histidine auxotrophs [147] and for the induction of a suppressor (supQ) of leucine auxotrophy {leuD) [133]. Many such studies are concerned primarily with genetic phenomena other than mechanisms of mutagenesis. For example, DES has been used in a study of reversion of auxotrophies in a highly mutable strain of Bacillus pumilus [176]. DES has, in addition, been used for mutagenesis in a number of bacteria which are employed less frequently in genetic studies. Among these are Pseudomonas aeruginosa [151], Streptococcus lactis [333], Staphylococcus aureus [49], Brevibacterium ammoniagenes [202], and Actinomyces rimosus [201].

Mutagenicity in transforming DNA That the dialkyl sulfates inactivate transforming DNA was demonstrated for Haemophilus influenzae as early as 1956 [344]. At equal concentrations, DMS was much more strongly inactivating than DES [344]. This result is in accordance with the relative alkylating activity of the two compounds and with the nature of the alkylations. Among the most widely used tests for mutation-induction in transforming DNA are those based upon the tryptophan operon and a closely linked marker for histidine auxotrophy in Bacillus subtilis [121]. Several variations of this system have been used to determine mutation frequencies for fluorescent, indole-requiring mutants [38,121]. Transforming the tryptophan auxotrophic strain T3 of Bacillus subtilis to tryptophan prototrophy and scoring for fluorescent, indole-requiring mutants in medium containing suboptimal amounts of indole, Bresler at al. [38] have shown DMS to be mutagenic in transforming DNA. The mutation frequency was shown to increase in a dose-dependent manner, and at the highest exposure, fluorescent mutants constituted 2% of total transformants.

96 Although distinctly active, DMS is not a very potent mutagen for transforming DNA when compared to hydroxylamine, nitrous acid or hydrazine [38]. When m u t a n t frequency is plotted against the average number of lethal hits per unit DNA length, all these agents yield linear responses [68]. The slope of the line for DMS, however, in only 1/7 that of hydroxylamine and 1/3 to 1/2 that of nitrous acid and hydrazine. Mutant frequencies as high as 10% were reported for hydroxylamine. Of the mutagens tested, only UV was less effective than DMS in terms of mutants per lethal damage. The strong inactivation of transforming DNA caused by DMS is probably attributable to strand breakage resulting from alkylation. It has been suggested by Bresler et al. [39,40] that inactivation occurs largely by the shortening of recombination lengths of DNA due to nonspecific DNA lesions. Consistent with this hypothesis, inactivating effects of several different agents used in combination are found to be additive regardless of their molecular mechanisms of action [39]. Among the agents for which such additivity was observed are DNAase and several mutagens, including DMS [39]. Evidence has been presented [41] that the inactivating lesions induced b y DMS in transforming DNA are subject to excision repair. Transforming activity for the trp3 marker was found to be greater for DMS-treated DNA in a repair-proficient recipient than in hcr- or uvr- repair-deficient strains. Mutagenicity in blue-green algae The first report of mutagenesis by DES in a blue-green alga involved the induction of streptomycin-resistant mutants in Anacystis nidulans [107]. Subsequently, penicillin-resistant mutants were recovered in Anabaena variabilis mutagenized with DES or with DMS [109]. Penicillin-resistance in Anacystis nidulans and streptomycin-resistance in Anabaena variabilis were also selected following DMS treatment. The recovery of a chlorophyll- and carotenoid-deficient mutant in Anacystis following mutagenesis by DES [108] has been described. Recombinogenicity in bacteria The recent studies of Norin and Goldschmidt [223] provide evidence that recombinogenic effects of DES may be detected in bacteria. The measure of recombinogenicity used was nonreciprocal recombination between the hisC locus on an F' episome and that on the E. coli chromosome. DES, as well as EMS, MMS, and MNNG, was found to stimulate the rate of occurrence of this recombinational event, which is analogous to induced mitotic gene conversion in yeast. Bacterial repair assays I t has been shown by Slater et al. [289] that the E. coli polA- strain P3478 is more sensitive to a variety of DNA-damaging agents than is the wild-type strain E. coli W 3 1 1 0 from which it was derived. The greater sensitivity of the poIA strain is attributable to the involvement of DNA Polymerase I in the process of excision repair. A mechanism underlying the greater sensitivity of polA strains {they do excise damage) has been proposed by Cooper and Hanawalt [58]. Moreover, measuring the differential sensitivity of the poIA ÷ and

97 p o l A - strains to killing in a spot test has been suggested by Slater et al. [289]

as a rapid method to screen for potential mutagens and carcinogens [289]. DMS was shown to be active in this test [83]. For DMS, MMS, and EMS, p o t e n c y in the p o I A test appeared to be proportional to total alkylating activity: DMS MMS > EMS [83]. DES was n o t tested. The data would suggest that at least some of the pre-lethal damage induced by methylating agents is subject to repair processes involving DNA polymerase I. A comparable repair assay has been proposed for Salmonella using the repairproficient strain TA1978 and strain TA1538 which contains a deletion through the uvrB gene and is consequently deficient in excision repair [15]. Unfortunately, this test is insensitive to some intercalating mutagens and some methylating and ethylating agents [15]. DES was found to be essentially negative in this test -- the diameters of the zones of inhibition being 15 mm in T A 1 5 3 8 and 14 mm in TA1978 [15]. Another repair assay which may be performed as a spot test in Petri dishes is based upon relative killing of wild-type Proteus mirabilis and a rec- hcr- strain in which repair of single-strand breaks is greatly reduced [4]. DMS was found to be active in the Proteus repair assay. The zone of inhibition formed by 0.001 ml of DMS was 23 mm in the wild-type strain, 29 mm in a rec- strain, and 31 mm in the rec- hcr- double mutant [4]. The two cyclic sulfuric acid esters PSF and BSF have also been reported as active in the Proteus repair assay [4,37] while a third cyclic sulfate, ESF, was n o t [37]. Since ESF is the most p o t e n t alkylating agent of the group and is extremely unstable in water [37], its inactivity may be ascribable to its instability. Greater killing of a rec- strain of Bacillus subtilis than of a comparable rec ÷ strain has been reported for DES [217]. The frequencies of auxotrophic mutants recovered following DES treatment were also reported to be higher in the rec- strain [217,241]. Prophage induction

The detection of prophage induction in lysogenic strains of bacteria has been suggested as a m e t h o d for identifying genetically active agents [116,194,206]. The most thoroughly studied system of this t y p e is the induction of the phage of E. coli [116,206], b u t other systems have also been used. The activity of DMS for prophage induction was reported by Field and Naylor [78] for a strain of Micrococcus lysodeikticus lysogenic for bacteriophage N5. Although the tests were not extensive, the activity of DMS appeared to be roughly equivalent to that of ultraviolet light and greater than that of ~-propiolactone. The activity of DES for phage induction has been demonstrated in E. coli strain P4X6, lysogenic for lambda [194]. The method employed was a spot test in which DES was applied to an antibiotic assay disc on a lawn of lysogenic cells. A ring of plaques in the lawn surrounding the zone of inhibition caused b y the chemical indicated a positive test. The activity of DES for inducing prophage ~ has also been reported by Hussain and Ehrenberg [129] who found DES to be more effective than EMS b u t less effective than a number of other alkylating agents. No correlation between activity for phage induction and Svcain--Scott s-value was evident [129]. Phage induction by DES, as well as by

98 MNNG, mitomycin C, and ultraviolet light, has also been reported in a spot test with Acinetobacter lysogenic for phage P78 [120]. Genetic effects in fungi Induction of mutations

An early report of the genetic activity of dialkylsulfates in fungi was presented at the 1953 meetings of the Genetics Society of America by Kc,blmark and Giles [139]. The system used was reversion of auxotrophic strains of N e u r o s p o r a crassa. A few years later in a paper summarizing early results for chemical mutagenesis in Neurospora, Westergaard [332] presented data for the reversion of an adenine auxotroph, a d 3 8 7 0 1 , by a variety of compounds including DMS and DES. Although the response to DMS appeared to be greater than the response to DES [332], comparing mutagenic potencies on the basis of reversion frequencies may present some difficulties because a variety of phenomena other than the intrinsic mutagenicity of the compounds may contribute to observed frequencies. In addition, differences in toxicity of the two compounds complicate the comparison. K¢lmark [ 138] reported dose-dependent increases in reversion frequency for the a d 3 8 7 0 1 and i n o s 3 7 4 0 1 alleles in a strain of N e u r o s p o r a crassa auxotrophic for both loci. He found, however, the relative effectiveness of DMS and DES to be different for reversion of the two auxotrophic markers. For treatments yielding maximum mutation frequencies, the ratio of ad ÷ to inos ÷ revertants was 19 for DMS and only 4 for DES. Besides varying between compounds, such ratios also varied markedly for different treatment times with the same compound. Studying reversion of the same two alleles in Neurospora and the ad6-45 allele in Saccharomyces, Marquardt et al. [192] obtained somewhat different results. They found dose-dependent increases in reversion frequency for the a d 3 8 7 0 1 allele but found i n o s 3 7 4 0 1 and the yeast ad6 mutation to be refractory to reversion by DES. In the same study, however, reversion of both these alleles by nitrosomethylurethane was observed. No mechanism was proposed to explain the apparent specificity. The reversion of adenine- and inositol-auxotrophic strains of N e u r o s p o r a crassa has received considerable attention in other studies pertaining to mutagen specificity [24,26--28]. It has been clearly pointed out that apparent specificity at the molecular level must be interpreted with caution, as the causes of such specificity may in some cases lie in physiological aspects of mutation expression rather than in primary interactions between mutagens and DNA. Mailing [187] has shown that DES can revert a majority of ad3 mutations induced by nitrous acid in N e u r o s p o r a crassa. The frequencies of reversion induced by DES, EMS, and nitrous acid were consistent with all 3 agents inducing predominantly base-pair substitutions [187]. Mutagen specificity in Neurospora has also been studied for reversion at the m t r (4-methyltryptophan resistance) and hist-2 loci and at a third locus which suppresses the m t r phenotype [42]. DES and EMS were found to be very similar to one another and different from UV, ICR-170, and MNNG in spectrum of mutations induced. While

99 the molecular mechanisms of reversion in this system are n o t clearly defined and some differences in interpretation of results are possible, the data are consistent with the suggestion that DES and EMS induce primarily base-pair substitutions but also a low level of frameshift mutations. Other studies in fungi with EMS [188] and DES [ 51] support the same interpretation. Among the mutagenicity studies conducted with Neurospora is a report involving DPS and DBS [138]. No mutagenic effect and essentially no toxic effect was observed with DBS. With DPS, however, revertants for ad and inos alleles were detected despite the absence of a reduction in survival. A m u t a t i o n system which is especially effective for evaluating the molecular mechanism of action of mutagenic agents is that developed in Saccharomyces by F. Sherman and his coworkers [239,267]. In this system revertants are selected in iso-l-cytochrome c (cy-1) mutants that have been biochemically characterized to the extent that the specific base-pair substitutions which can revert a particular allele are known. Prakash and Sherman [239] found that DES preferentially induces G - C to A • T transitions in the cy-1 system. DMS, however, did n o t exhibit the same specificity. Consistent with the results for the dialkylsulfates, EMS was quite specific for G • C to A • T transitions whereas MMS was not. Specificity for the G" C to A " T substitution was determined by relative revertibility of cy1-131 and other cyl alleles. The cy1-131 allele is known to revert only by a G • C to A • T transition. None of the other tester strains employed revert by this mechanism [239]. In the cyl system, a DES treatment causing less than 50% killing increased the reversion frequency for the cy1-131 allele from a spontaneous frequency of less than 1 m u t a n t per 107 survivors to 240 per 107 survivors [239]. In all other strains the reversion frequency was less than 10 per 107 survivors following this DES treatment. A DMS treatment which increased the reversion frequency in cy1-131 to only 5 per 107 survivors, however, produced reversion frequencies greater than 20 per 107 in several other cyl alleles. Prakash and Sherman [239] proposed that the greater mutagenicity and mutagen specificity of DES relative to DMS may be attributed to the greater efficiency of the former c o m p o u n d for forming O6-alkylated guanine residues in DNA. The data would furthermore be consistent with DES acting to a significant extent by the direct mispairing mechanism of mutagenesis and DMS acting by an indirect mechanism. While the cy-1 system permits relatively unambiguous determinations of mutagen specificity, Prakash and Sherman [239] point out that even in this system there are complexities of interpretation due to such phenomena as the effects of neighboring bases on frequencies of reversion of particular alleles. The data of Eisenstark et al. [75], discussed in the bacterial section of this review, would suggest, moreover, the need for caution in concluding t h a t DES is highly specific for G • C to A • T transitions. Evidence that DES can induce reversions in cyl strains with A . T at the m u t a n t site, although at a much lower frequency than in cy1-131, is found in the reversion of particular ochre (cyl-9) and amber (cy1-179) mutations by DES [301,302]. Only reversion with A • T base pairs at the m u t a n t sites could explain the particular amino acids found in the positions in revertant iso-l-cytochrome c corresponding to the nonsense codons in the mutants. The data indicate that DES induced both

100

A • T to G • C transitions and A • T to T • A transversions [301,302]. In the case of cyl-9, evidence was also found for an A • T to G • C substitution [302]. Results obtained with the two nonsense mutants suggest, in addition, that nearby bases can affect proportions of different substitutions induced [ 301]. The reversion of a large number of cy-1 mutations by DES and EMS has also been reported from qualitative studies using a spot-test m e t h o d [267]. The mutants responded to the two mutagens in essentially the same manner, suggesting c o m m o n mechanisms of action for both compounds [267]. Brusick and Zeiger [51], studying reversion in methionine auxotrophic strains of Saccharomyces which contain presumptive base-pair substitution and frameshift mutations, found DES to be active in spot tests with the substitution strain and marginally active in the frameshift strain. These results are consistent with those obtained in other systems to the extent that DES is an effective base-pair substitution mutagen. The methionine auxotrophic strains used [234], however, are characterized only in terms of the agent by which t h e y were induced and their pattern of revertibility. They do not appear to be sufficiently well characterized, therefore, to distinguish whether the weak activity of DES in the frameshift strain is due to incomplete specificity of the chemical or low levels of reversion by mechanisms other than frameshift mutations. Fink and Lowenstein [80] have also reported the induction of reversions by DES in auxotrophic strains of Saccharomyces. Effective mutagenesis occurred when DES was added to plates containing his-4 mutants in rich medium, and the yeasts were replicated after one day onto medium selective for revertants. The mutagenicity of DMS [125] and of DES [123,125] in the yeast Schizosaccharomyces pombe has been demonstrated by Heslot. The induction of forward mutations by DES [123] was detected at the ad-1, ad-3, ad-4 and ad-5 loci for which mutants can be scored as white colonies arising in a red ad-7 mutant strain. Dose-dependent increases were detected in the frequency of prototrophic revertants in arg-1, ur-1 and leu-3 mutants treated with DES [125]. The m a x i m u m reversion frequency reported for DES was similar to that for EMS and higher than that found for a variety of other mutagens [125]. A dose-dependent increase in the frequency of reversions at the arg-1 locus of Schizosaccharomyces pombe has also been reported for mutagenesis by DMS [125]. If m u t a t i o n frequencies are compared for a given chemical concentration, DMS is of greater mutagenic activity than DES [125]. If such comparisons are made at equal levels of survival, however, DES is more highly mutagenic than DMS. As in a number of other studies, the data of Heslot [125] show similarities between DMS and MMS and between DES and EMS. In Aspergillus nidulans, a methionine auxotroph (meth-1) which reverts to p r o t o t r o p h y by suppressor mutations in at least 5 and possibly 7 different loci has been employed in a number of mutagenesis studies [171,255]. Up to 1000fold increases in m u t a t i o n frequency have been reported for DES treatments at survival levels between 1 and 10% [8]. Increases in m u t a t i o n frequency of about 70-fold have also been reported for DMS treatments at about 45% survival [209]. There is apparent interlocus specificity in the m e t h , suppressor system in that the proportions of the different classes of mutants are different among DES-induced mutations, EMS-induced mutations, and spontaneous mutations [8,209]. The mechanism underlying this apparent specificity, however, is uncertain.

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Another mutation system in which the mutagenicity of DES has been studied in Aspergillus nidulans is that based upon conidial color and xanthine dehydrogenase (XDH) activity [9,11]. In the presence o f 2-thioxanthine, the wild-type produces yellow conidia instead of the normal green. Mutants with altered XDH activity are detected by their production of green conidia in the presence of the purine analogue. Such XDH mutants may be assigned to at least 8 genetic loci. There are, in addition, mutants resistant to the pigmentation effect of 2-thioxanthine b y mechanisms other than XHD-deficiency [9,11]. DES has been shown to be effective for inducing forward mutations at a number of loci in this system [11]. As in the meth-1 system, mutagens differ from one another in the distribution of mutations among the loci [9], but the meaning o f the differences remains to be determined. DES has also been shown to induce reversions of XDH mutants in Aspergillus nidulans [7,113]. Revertants are selected by plating conidia on media in which hypoxanthine is the sole nitrogen source. The suitability of the XDH system for specific revertibility tests is questionable, however, because mutants induced by ICR-170 are as readily reverted by DES and MNNG as by ICR-170

[7]. In Aspergillus nidulans, Hartley [112] has studied the induction of reversions by DES in nia-D mutants which are unable to use nitrate as their sole nitrogen source. A curiosity reported in this study is the rather extreme resistance of one nonrevertible strain, niaD-14, to the killing effect of DES but not to that of MNNG. While the mechanism underlying the DES-resistance is unknown, some interesting speculation on the topic has been published [ 112]. Another fungus in which the mutagenicity of dialkyl sulfates has been studied is the ascomycete Ophiostoma multiannulatum in which DMS was used to induce reversions in several auxotrophic strains [345]. The utilization of DES to mutagenize strains of Saccharomyces vini for the purpose of obtaining variants useful in wine production has also been reported [12]. Similarly, D~ES has been used to select for variants of interest in other fungi such as Eremothecium ashbyii [300]. A report of the induction of chromosomal rearrangements in fungi may be found in a paper by Alderson and Hartley [9] who cite the doctoral dissertation of B.M. Rever as showing that DES induces translocations in Aspergillus

nidulans. Induction of mitotic recombination A strong correlation has been established between mutagenicity and recombinogenicity in yeasts. Mutagens of a diversity of molecular mechanisms o f action have been shown to induce mitotic recombination of both the reciprocal (mitotic crossing-over) and nonreciprocal (mitotic gene conversion) types [347--350]. The induction of mitotic crossing-over b y DES was reported by Zimmermann et al. [350] for strain D1 of Saccharomyces cere~isiae. For a treatment with DES causing very little cell killing, a frequency of 8.'9 × 10 =3 was observed for mitotic recombination as measured by the ad2 and hi8 loci. The control frequency was less than I × 10 -4. The strain used most extensively for detecting mutagen-induced mitotic gene

102 conversion is strain D4 of S a c c h a r o m y c e s cerevisiae. This diploid strain is heteroallelic at the ad2 and trs loci and provides a positive selection for ad ÷ or trp ÷ convertants [346,347]. The activity of DES in strain D4 was demonstrated by Zimmermann [346] who reported 10- to 15-fold increases in convertant frequencies from a b o u t 1 X 10 -s per surviving cell. The work of Zimmermann [346] has also provided some indirect evidence for repair of DES-induced damage in Saccharomyces. Respiratory-deficient cells on a nonfermentable substrate exhibited reduced survival and higher convertant frequencies following a period of post-treatment liquid holding. A 2-day liquid-holding period increased the convertant frequency at both loci by more than 2-fold and decreased survival to a b o u t 65% of that without liquid holding. Respiratory-competent cells, on the other hand, underwent liquidholding recovery. The data suggest that normal repair of DES-induced damage is inhibited by the inavailability of respiratory energy. Genetic effects in vascular plants Induction of mutations

The scientific literature pertaining to the treatment of plants with DES is extensive. The primary objective in many of the studies is introduction of genetic variability into the plant species rather than investigation of the mutational processes involved. Since it is n o t m y intent to review this aspect of mutagenesis, my comments will be largely restricted to papers directly concerned with the nature of mutagenesis in plants. Several systems which have been used for mutagenicity studies in plants have been reviewed by Ehrenberg [72]. Among plant systems, the detection of chlorophyll mutations in grasses has been used most extensively in studies involving DES. The reported frequencies of chlorophyll mutations depend heavily on h o w those frequencies are expressed. For example, the chlorophyll m u t a n t frequency reported for rice "seeds" (caryopses; one-seeded fruits) treated for 2 h with 0.076 M DES was 59.6% when expressed per plant in the treated (M1) generation, 21.4% when expressed per M~ panicle, and 2.3% when expressed as a proportion of seedlings in the second (M2) generation [242]. The corresponding control values were 0.60, 0.20, and 0.01%, resp. There is some disagreement as to the relative merits of these 3 means of expressing m u t a n t frequency, and the problem has been discussed by Rao [245]. While the effects of DES have received considerable attention, the number of systematic studies involving DMS in plants is much more limited. DMS has been reported to be active in inducing chlorophyll mutations and reducing numbers of fertile spikelets in barley [72,73,126]. The frequency of mutations recovered, however, is generally appreciably less with DMS than with DES [126]. Negative results have also been reported for DMS in the barley chlorophyll system [17,221]. Besides barley, the induction of chlorophyll mutations by DMS has been reported in wheat [189] and Arabidopsis [236]. The mutagenic action of DES has been studied extensively in barley [63, 119,126,144,221,243,245]. Dose-related increases in frequencies of chlorophyll mutations occur following exposure of "seeds" to DES [96,119,144]. According to Nilan et al. [221], DES and EMS are among the most p o t e n t mu-

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tagens known in the barley system. A maximum frequency of 43 chlorophyll mutations per 100 MI spikes has been reported for a hull-less variety of barley treated with DES [221]. The comparable frequencies for 7-rays and nitrogen mustard are 17 per 100 and 15 per 100, resp. [221]. A number of earlier studies [119,124,126,144,221] also demonstrated the induction of chlorophyll mutations in barley by DES but at lower frequencies. The difference is ascribable to the development of more effective treatment conditions [221]. In the studies of Doll and Sandfaer [63], dose-dependent increases were observed in sterility of treated plants and in the frequency of chlorophyll mutations in the second generation. "Seeds" were treated for 5 h with aqueous solutions of DES at concentrations from 0.08 to 0.2%. The frequency of chlorophyll mutations increased from a control value of 0.04% to as high as 4% at the highest dose of DES. Morphological mutants were also scored and were found to increase in frequency proportionally with the chlorophyll mutants. In terms of mutant frequency per unit sterility, DES was a more efficient mutagen than 7-rays and approximately the same as EMS. In addition to barley, the induction of chlorophyll mutations by DES has been studied in rice [34,54,101,218,242,248,293,315], wheat [36,62,215], sorghum [294,296--298], fodder peas [204,205], foxtail millet [105], and peas [197]. As in barley, DES is a more efficient mutagen than X-rays in terms of the frequency of chlorophyll mutations induced in fodder peas at similar levels of survival [204]. In both wheat [62] and foxtail millet [105], dose-dependent increases in mutation frequency were detected for chlorophyll mutations per M I spike or as a proportion of M2 seedlings. There were apparent differences between wheat varieties with respect to sensitivity to DES [62]. Based upon differences in pigmentation, chlorophyll mutants may be classified into several categories, such as albina, viridis, xantha, and striata. It has been pointed out in several studies that the relative frequencies of these mutant classes differ for different mutagens. Among the agents for which such apparent specificity has been reported is DES [34,54,63,119,144,299,341]. The spectrum of mutations detected may vary not only from one mutagen to another but also with differing treatment conditions [341]. The molecular explanation for specificity with respect to the different classes of chlorophyll mutations is obscure. Another system that has been employed for the study of mutagenesis in plants involves selection in oats for resistance to the toxin produced by Helminthosporium victoriae [331]. Such resistance is conferred by genetic alterations, probably point mutations and/or deletions, at the Vb locus. When "seeds" are soaked in solutions of DES, dose-dependent increases Occur in the frequency of resistant M2 seedlings [331]. As shown in Table 9, morphological mutations have been detected in a number of the same plant species used to study chlorophyll mutations [36,54,63, 106,204,205,215,247,297,299,315,341]. The induction of sterility by dialkyl sulfates has also been recorded in a number of studies. For example, dosedependent reductions in fertility have been reported for sunflowers treated with DMS [235]. Similarly, reduction in fertility is reported for wheat treated with DES [62]. The proper genetic interpretation of induced sterility is uncertain, however, because the mechanism by which it occurs appears to differ

104 from one mutagen to another [76]. Sterility induced by ionizing radiations frequently involves chromosome aberrations whereas that induced by chemical mutagens may n o t [76]. In view of the relatively low efficiency reported for induction of chromosome aberrations by DMS and DES, it has been suggested that the induction of sterility by these agents might be attributable to mutations [119,144]. The induction of mutations in quantitative characteristics is of particular interest for producing variability to be used in plant breeding programs [47]. While radiations have been used more extensively than chemical mutagens in this regard, data for chemicals, including DES, are available. Povilaitis [237] has reported mutagenic effects of DES on quantitative characteristics in tobacco. Dry seeds were soaked in a saturated solution of DES for 3--5 h. Seedlings which appeared normal (designated C1 generation) were used to produce a second (C2) and third (C3) generation by self-fertilization. Populations derived from treated plants exhibited smaller mean plant heights and leaf lengths and later mean flowering times than did the controls. Significantly, the variances and ranges of variation were higher for all treated populations than for untreated. The induction of genetic changes in quantitative characteristics by DES has also been reported for Arabidopsis thaliana [45,47]. Changes in variances, means, skewness, and kurtosis with respect to flowering time and plant weight were reported for the M2 generation derived from plants which had been mutagenized as seeds [47]. The dialkyl sulfates have been used extensively for the purpose of obtaining genetic variants in plants. These efforts involve a large number of plant species and especially plants of economic importance. Variations studied in mutagenized plants and their descendants include a broad variety of morphological characteristics as well as a number of physiological characteristics such as growth rates and flowering times. Of primary interest in a number of the studies are attributes of agricultural significance. The data in the files of the Environmental Mutagen Information Center, Oak Ridge, TN, reveal that DMS has been used for the purpose of mutagenesis in at least 40 different genera of vascular plants. DES has similarly been used on plants of at least 35 different genera. Among the studies involving induced variations in plants are some, such as those of Ashri [19] with peanuts, in which the pattern of inheritance of the variations was analyzed over a period of several generations. In other studies, however, variants were reported b u t their genetic basis was n o t analyzed. Similarly, in some of the studies [247] there is evidence of dose-dependence in the recovery of mutants whereas in others either only one dose was used or there was no evidence of dose-dependence. Among the approx. 100 plant species that have been mutagenized with dialkyl sulfates are species of oats [268], wheat [146,190], rice [170,247], sweet peppers [288], fodder peas [204], sunflowers [235], apple [140], cowpeas [265], Arabidopsis [111], peanuts [20], soybeans [57,186], peas [197], barley [150], pearl millet [52], and maize [270]. Evidence that chemically mutagenizing agricultural plants can have practical benefit is provided by the commercial growth of varieties of barley containing variations introduced by treatment with DES [222].

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Induction o f chromosomal alterations A number of alkylating agents, including the dialkyl sulfates, induce chromosome breakage in the cells of plants and animals. In general, the ratio of chromosome aberrations to point mutations is higher for polyfunctional alkylating agents than for monofunctional agents [22,71,221]. Accordingly, the dialkyl sulfates are often reported to be somewhat inefficient as clastogenic agents in comparison to their mutagenicity. Preliminary data for DMS [119] suggest that this compound is not an effective inducer of chromosome aberrations in barley. Dose-dependent increases in the frequency of anaphase bridges and fragments have been reported, however, in mitotic cells of sunflowers [235]. Following a 24-h treatment of seeds with DMS at concentrations of 0.02, 0.04 and 0.05%, root-tip squashes revealed that 2.0, 5.0 and 6.9% of cells, resp., contained aberrations; the control value was 0.7%. Dose-dependent increases in the frequency of chromosome aberrations following treatment with DMS have also been reported in wheat [269]. Although 12--24-h treatment times were used and hydrolysis of the chemical during treatment was apparently not considered, aberration frequencies as high as 46.6% were recorded; the control frequency was 1.4% [269]. Two other plants in which DMS has been reported to induce chromosome aberrations are Vicia faba [173,309] and the Norway spruce [320]. As in the case of mutagenic effects, DES has been more thoroughly studied for clastogenic effects than has DMS. Although active, DES is found to induce few chromosome aberrations relative to its highly efficient induction of chlorophyll mutations [119,144,221]. There are a number of papers describing the induction of chromosomal alterations in barley by DES [94,95,119,144,199,200,220,243,244]. Rao [243] has reported dose-dependent increases in the frequency of mitotic anaphase chromosome fragments and dicentric chromosome bridges. Frequencies of 2.9 bridges and 12.5 fragments per 100 cells were found for DES-treated plants while no aberrations were found in the controls. The frequencies reported for X-rays at the same level of plant killing (11%) were 6.4 bridges and 30.4 fragments per 100 cells [243]. Similarly, Mikaelsen and coworkers reported an increased incidence of fragments and bridges in shoot-tip cells [200] and of acentric fragments in root-tip cells [199] following treatment of barley "seeds" with DES. Low levels of induction of chromosome aberrations by DES have been found for meiotic as well as for mitotic cells of barley [144]. The meiotic alterations include translocations, but the efficiency of induction is very low compared to that of ionizing radiation [ 144]. The induction of chromosome breakage by DES has been reported in several other species. Among these is Crepis capillaris for which the data, although not extensive, support the conclusion that, in comparison to 7-rays, DES is extremely inefficient for the induction of aberrations in root-tip mitotic cells [119]. Data from Vicia faba, in which the alterations detected were primarily of the chromatid type, support the same conclusion [117]. In Bellevalia romana the distribution of chromosome breaks induced by DES was found to be distinctly non-random, and chromosomal rearrangements were not observed [98]. Differences were recorded between DES and other agents both in terms of the distribution of breaks and in the occurrence of rearrangements. The

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general conclusions to be drawn from the differences are, however, obscure [98,99]. Other plants for which there have been reports of chromosomal alterations induced by DES include tomato [35], sorghum [295], Nigella damascena [233], and Cuminum cyminum [214]. Another cytogenetic effect that has been observed in plants following exposure of seeds to DES is chromosome loss from trisomics. The percentage of M~ generation plants carrying the extra chromosome in root-tip cells was reduced to 4% from a control value of 34% in a strain of barley trisomic for chromosome-4 [258]. A similar reduction from 28 to 9% occurred in a trisomy-5 strain [258].

Induction o f paramutational changes The phenomenon of paramutation has been described in the classic studies of Brink and his coworkers [30,43,44]. The term paramutation refers to heritable but genetically unstable changes that occur at high frequency under the appropriate conditions. While such changes are observed in several plants [44], they have been studied most extensively at the R locus in maize which controls anthocyanin production in seeds. The ability of DES to induce paramutational changes in maize has been demonstrated by Axtell and Brink [30]. The levels of induced alterations were very high compared to frequencies characteristic of mutation induction in that more than 10% of MI generation plants treated with DES exhibited paramutational changes [30]. The alterations induced by DES were shown to be heritable [30]. Brink [30] has proposed that paramutation may involve repetition of a regulatory sequence near the R locus. Mutagens have been shown to stimulate the formation and loss of genetic duplications in bacteria [127,128] and could have similar effects in plants. Whatever the explanation, paramutation represents an intriguing genetic phenomenon inducible by DES. Factors modifying response to mutagens A number of factors associated with treatment conditions have been shown to affect the response of plant systems to mutagens. Such factors may alter the induction of mutations, per se, or may affect mutagenic efficiency through modifications in toxicity. Among these factors are the half-life of the compound under the treatment conditions [143,221], the concentration of hydrolysis products [143], and the relationship between concentration and duration of treatment [143,221]. Specifically, high concentrations of DES increase seedling injury and killing disproportionately to mutation frequency [143]. Mutagenic efficiency is greater for longer treatments at low concentrations [143]. Such treatments require the replacement of DES solutions to correct for hydrolysis of the compound. Seed size [143,221], whether seeds are dry or soaked [111,131,200,221,244,298,315,340], whether they are stored following treatment [96,143,200,216], and whether they are hulled or hull-less [111, 131,143,221] are other factors which affect the yield of mutants. Temperature and oxygen supply can also modify the response of plants to mutagenesis by DES [118,143,145,221]. Possible modifying effects of pH are particularly noteworthy because of the highly acidic hydrolysis products of DES [170,200, 221,244,335]. In general, the hydrolysis products of DES do not appear to be

107 mutagenic, but pH may alter mutagenic efficiency through differences in toxicity. Cell-cycle stage during treatment is an additional factor which may affect the yield and spectrum of mutants [131,315,316,339--341]. Similarly, the developmental stage of the embryo in treated seeds can substantially alter sensitivity to DES [21]. The presence of dimethyl sulfoxide (DMSO) along with DES in the treatment solution has been reported [218] to cause an increase in the frequency of chlorophyll mutations in the M2 generation of rice relative to that induced by DES in water. The opposite result has, however, also been published [18]. It is perhaps noteworthy that DMSO has been found to enhance the toxicity of EMS [315]. It is likely that modification by DMSO of mutant recovery following treatment with DES is a consequence of toxicity modifications and may involve greater solubility of DES or its improved permeation into cells. An interesting observation regarding the effect of pre-treatment seed soaking in barley is that, while soaking seeds for 17 h increases the frequency of chlorophyll mutations recovered relative to soaking for 5 h [199,200], the reverse was observed for the frequency of chromosome fragments [199]. There is some evidence that cell-cycle stage during treatment is an important factor in the induction of aberrations. Mikaelsen [199] has suggested in this regard that G I cells are most sensitive to DES and EMS whereas G2 cells are most sensitive to 7-rays. Studies involving synchronized cells or accounting for possible effects of alkylating agents on the duration of the cell cycle were not, however, conducted. That such effects could influence results is suggested by the work of Heiner [117] showing that DES causes a dose-dependent delay in the mitotic cycle of Vicia faba root tips due to blockage in the S-period of interphase. The data of Heiner [117], moreover, are most easily explained by the S-period being the part of the cell cycle most sensitive to the chromosome-breaking effects of DES. The data of Yamaguchi [340] would similarly indicate that the S-period is most sensitive to mutation induction by DES. The data of Heiner [117] and Mikaelsen [199] are in agreement that G2 cells are relatively insensitive to DES-induced chromosome damage. As with chlorophyll mutations, the yield of chromosome aberrations induced by DES is also sensitive to other factors in the treatment conditions, including pH [200] and post-treatment seed storage [94--96]. According to Gichner and Velemfnsk:~ [94,95], aberrations induced by treatment of barley seeds with DES are mostly of the chromatid type, and post-treatment seed storage results in a shift toward preferential induction of chromosome-type aberrations [94,95]. The authors suggest that the change may be ascribable to preferential repair of lesions leading to chromatid-type aberrations and conversion of some of these lesions into chromosome-type aberrations [95]. Relative to DES treatment with no post-treatment seed storage, the total frequency of aberrations is reported to be increased by storage of seeds at 20% water content [94] but decreased by storage at 30% water content [95]. Possible interactions among mutagens There have been several reports of increased efficiency in obtaining mutants by the use of mutagens in combination [63,242,266]. Among the mutagens for

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which synergistic interactions have been proposed to occur is DES [197,242, 248,331]. Synergism has been reported [248] between DES and hydrazine and between DES and hydroxylamine for the induction of chlorophyll mutations in rice. More than additive effects were also recorded for reduction in fertility of seeds [248] derived from selfed M~ generation plants. Synergistic interactions have also been reported between DES and thermal neutrons for mutagenesis in rice [242]. In barley, Nilan et al. [220] reported synergistic effects of DES and X-rays with respect to frequencies of mitotic anaphase fragments. These results have been confirmed by Rao [243] who reported synergism between X-rays and DES with respect to seedling injury and mitotic anaphase chromosome fragments and additive effects for anaphase chromosome bridges and chlorophyll mutations in the M2 generation. In oats, Wallace [331] reported synergistic interactions between DES and ~,-rays for induction of mutations at the Vb locus, conferring resistance to Helminthosporium victoriae toxin. The data reveal mutation frequencies which are markedly higher than additive. Comparable synergism was not observed in measurements of germination percentage or seedling height. Wallace [331] has suggested that synergistic effects on germination and growth would also be expected if the large increases in frequency of toxin-resistance were ascribable to chromosome aberrations. The results were therefore interpreted in terms of synergism in the induction of point mutations [331]. The possibility that the mutants actually contain deletions or other alterations at the chromosomal level has not, however, been rigorously excluded. In contrast to these studies, a number of investigations of possible synergism among mutagens revealed no evidence for such interactions. For example, the data of Doll and Sandfaer [63] are consistent with the effects of "y-rays and DES being additive for the induction of both sterility and chlorophyll mutations in barley. The only case in which effects were found to be greater than additive was in reduction of the level of survival in the treated generation [63]. Similarly, Rao [242,243] found synergism between DES and X-rays for seedling injury in barley but additivity for chlorophyll mutations in the Ms generation. Not all reported interactions among mutagenic treatments in plants involve enhanced response. In the barley chlorophyll system, for example, it has been reported [142] that effects are less than additive when DES treatment is followed by treatment with sodium azide, another potent mutagen in this system. The opposite effect was observed for treatment with MNU followed by azide [142]. The basis for the interaction between DES and azide is unclear but may be related to increased physiological damage caused by the particular treatment used [142]. Less than additive induction of chlorophyll mutations by combined treatment of sorghum with DES and ~/-rays has also been reported [296]. An interesting report pertaining to interactions among mutagens in plants is that the effects of "y-rays and EMS in barley are less than additive for chlorophyll mutations expressed per M1 plant but more than additive when expressed per M~ spike or per M2 plant [1]. Such results suggest the possibility that apparent cases of synergism in plant systems may be artifactural, or at least

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may be more a consequence of the system than of interaction between mutagenic treatments, per se. Evidence for differences in apparent interactions attributable to different methods of expressing mutant frequency has also been found b y Rao [245] who has discussed possible explanations for such effects. Although the data do not unequivocally show either additivity or non-additivity of effects for combined treatments with DES and ionizing radiations in the barley chlorophyll system, they would seem to be most consistent with the absence of true interactions [245]. They illustrate, moreover, the need for careful analysis of the causes of non-additivity of mutagenic effects before conclusions are drawn with respect to synergisms. Distinct differences in effects of combined treatments at different mutagenic doses further complicate interpretations [245]. In summary, the general nature of possible interactions between DES and other mutagens in plants is unresolved. At present, the evidence for synergistic interactions seems stronger for the induction of chromosome breakage than for mutation induction [243]. Several explanations have been proposed to account for cases where interactions between mutagens have been reported [248]. In view of the inconsistencies in the plant literature as relates to synergism, however, generalizations must be regarded as questionable. This is particularly true because large differences exist in detection of mutants under differing experimental conditions and with different methods of analyzing mutation frequencies. Genetic effects in insects

Induction o f mutations in Drosophila The first report of the mutagenicity of dialkyl sulfates was that made by R a p o p o r t [246] who used the C1B method to demonstrate the induction of sex-linked recessive lethals in Drosophila. DES was administered in the food, and 2.4% lethals and a few visible mutations were detected. DMS was also found to be mutagenic, producing lethal mutations in 1.3--3% of treated chromosomes [138]. Since Rapoport's work in the early days of chemical mutagenesis, a number of additional studies have confirmed and extended the results on the mutagenicity of DMS and DES in Drosophila (Tables 8 and 9). One of the major advantages of Drosophila in mutation research is that in Drosophila it is possible to assay for a broad range of genetic effects in a relatively simple whole animal system [3,330]. Detectable genetic alterations include visible mutations and recessive lethals, which may be ascribed to point mutations or small deletions; reciprocal translocations; mitotic recombination; nondisjunction; chromosome loss; and dominant lethals [330]. Unfortunately, data for DMS and DES in Drosophila are not available for all these effects. Among Drosophila tests, that for sex-linked recessive lethals is the best validated and most extensively used [330]. It is a sensitive test in which high frequencies of recessive lethals, equivalent to approximately one mutation per genome [330], may be induced by DES. Dose-dependent mutagenic responses are observed following feeding with DES at concentrations ranging from 6.5 to 32.5 mM [228,330]. Alderson [ 5], using the Muller-5 (Basc) method, reported that at comparable

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levels of survival DES is a b o u t 5--6 times more effective than DMS for inducing sex-linked recessive lethals. For untreated flies, 0.25% of X-chromosomes scored carried lethals. For flies exposed to DMS by the adult-feeding method, the frequency of lethals was 2.11%. A comparable treatment with DES yielded 11.85% lethals. Survival values for the control and two chemical treatments were 98, 78 and 84%, resp. Pelecanos and Alderson [230] have shown that the induction of sex-linked recessive lethals b y DES may be detected when the c o m p o u n d is administered by larval feeding as well as by adult feeding. The dose-dependence of the response, however, was somewhat unusual in that a step-like increase in mutation frequency was observed at a DES concentration of 0.35--0.4% [230]. Frequencies of sex-linked lethals as high as 11.6% for male larvae and 15.7% for female larvae were induced by larval feeding with 0.5% DES. By the adult-feeding method, the high frequency of 27.1% lethals was reported for the same DES concentration. For 0.3% DES, the lowest dose tested, the frequency of lethals was 4.6% by adult feeding and 1.6--1.8% by larval feeding. The control value was 0.25%. It was suggested by the authors that the markedly lower mutagenicity observed for DES at concentrations below 0.35% administered by either m e t h o d of feeding is attributable to major components of the feeding mixture reacting with the mutagen and effectively reducing its concentration. The observation that excluding heat-killed yeast, the major food constituent, from the medium removed the threshold effect provides some support for this explanation. When adults were fed medium lacking the yeast, higher mutation frequencies were reported at the lower doses of DES, and the dose--response was essentially linear [230]. Differences among cellular stages with respect to sensitivity to mutagenesis by DES have been studied with the sex-linked recessive lethal test in Drosophila [10]. By the adult-feeding method, mature spermatozoa were found to be more sensitive than earlier stages in spermatogenesis. A progressive decrease in sensitivity was observed for spermatids, secondary spermatocytes, primary spermatocytes, and spermatogonia. Some differences in sensitivity were also reported for different cell stages in the immature testis of the larva when DES was administered b y the larval-feeding method. In adult females, the primary o o c y t e {Stage 7 o o c y t e ) and the mature unfertilized egg (Stage 14 o o c y t e ) were found to be of approximately equal sensitivity to mutagenesis by DES [231]. The sensitivity of these two stages, moreover, was somewhat greater than that of the intermediate Stages 8--13 [231]. Evidence for delayed mutagenesis by DES in Drosophila is found in the data of Alderson [6] who demonstrated an increase in the frequency of mosaicism for sex-linked lethals in the F1 produced by males treated with DES or other alkylating agents. Germinal mosaicism for recessive lethals in the FI was detected by the occurrence of both lethal and non-lethal cultures in an F3 produced by females of apparently non-lethal F2 cultures. The frequency of lethal mosaicism was 16.3% in the F1 produced b y DES-treated males compared to only 1.4% in the control. By carrying the experiments to an F4 generation, it is possible to detect lethal mosaicism in F2 females. No elevated levels of lethal mosaicism in the F2 attributable to DES or other mutagens was found. The occurrence of germinal mosaicism in the FI, however, suggests delayed effects

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in which recessive lethal mutations induced by DES are n o t immediately fixed in the zygote produced b y fertilization involving treated sperm [6]. Earlier data on the induction of sex-linked recessive lethals by DES have recently been confirmed by Mufioz and Barnett [210] who reported 27.1% {138/509) lethals following treatment of males with 0.5% DES. Another demonstration of sex-linked lethals induced by DES is that of Khan [134] who reported frequencies of 7.7--8.9% for complete lethals and an additional 7.1-7.3% for F1 mosaic lethals when adults were fed 0.5% DES. In a parallel study in which flies were fed 0.15% EMS, EMS was reported to be more active than DES for inducing recessive lethals [134]. The EMS treatment was, however, at lower survival. Similarly, Khan [135] has reported DES to be less effective as a mutagen in the recessive lethal test than is MMS. It has recently been demonstrated that recessive lethals may be induced by exposing adult males to DES as a vapor [2]. Frequencies of lethals as high as 26.7% are reported for treatment of spermatozoa. Consistent with the results from feeding experiments, lower frequencies were induced by exposures corresponding to earlier stages in spermatogenesis [2]. In addition to lethals, sex-linked visible mutations have been detected in Drosophila following treatment of adult males with DES [230]. The tests are, however, not as effective as those for lethals and the frequencies detected were low [230]. They do provide information, nevertheless, regarding the origin of mosaics following the treatment of spermatozoa with DES [6,230]. Because of greater difficulty in detection, autosomal recessive lethals are less generally studied than sex-linked recessive lethals. DES, however, has been tested for induction of autosomal recessive lethals and was shown to be active [228]. When DES was administered by larval feeding, 53% of the flies tested yielded lethals and 102 out of 464 chromosomes scored {22%) contained lethals. An interesting discovery made by Pelecanos [229] is that the distribution of lethal mutations in chromosome 2 of Drosophila is highly non-random. When males were exposed to 0.5% DES b y the adult-feeding method, a lethal frequency of 25.4% was detected. Of the 38 lethats tested, all could be assigned to one or more of 3 relatively small, separated, chromosomal regions. Comparable data for the distribution of mutations induced by other agents were not presented, and the explanation for the rather extreme site-specificity of DESinduced mutations is obscure.

Induction of male crossing-over in Drosophila Although crossing-over does n o t normally occur in males of laboratory strains of Drosophila, it may be induced by mutagens [228]. When flies were fed 0.5% DES throughout larval life, 7 of 47 males exhibited crossing-over with respect to 3 linked genes on the second chromosome. In the control, no crossovers were recorded for 70 males. For DES-treated flies, 0.31% of gametes involved crossovers compared to none in the controls. Induction of chromosome rearrangements in Drosophila Based upon a comparison of the sex-linked recessive lethal test and other tests in Drosophila, it has been suggested [330] that DES is an effective mutagen which lacks or has slight chromosome-breaking activity. This suggestion is

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based, at least in part, upon data indicating that DES, even at high concentrations, yields negative results in a test for reciprocal translocations between chromosomes 2 and 3 [228,330]. For example, Pelecanos [228] detected no translocations in a total sample of 2515 gametes from males and females fed 0.5% DES as larvae or from males fed DES as adults. The method used was that based u p o n the markers for brown and scarlet eye color on the second and third chromosomes, resp. [3]. Contrary to the early reports, recent data would indicate that DES does, in fact, induce reciprocal translocation in Drosophila. Mufioz and Barnett [210] have found that when adult males are fed 0.5 or 0.75% DES, mated, and the treated sperm are then stored in untreated females, reciprocal translocations between the second and third chromosomes are detected in .the progeny. Consistent with earlier reports, no translocations were detected in flies derived from fertilized eggs deposited less than 6 days after treatment of the males. For sperm storage times longer than 6 days, however, translocations were detected. The frequency of translocations increased with storage time to a maximum of 5.8--7.9% of tested chromosomes after 23--29 days of storage. No translocations were detected in the controls. Storage longer than 29 days was not possible due to depletion of sperm. The sperm-storage effect is not totally unexpected. For several other mutagens, storage of sperm has been found to increase the frequencies of detected genetic alterations involving chromosome breakage [ 134]. Among these alterations are translocations induced by nitrogen mustard [260] and by 2,4,6tri(ethyleneimino)-l,3,5-triazene [122] and small deficiencies induced by mustard gas [29]. Comparable storage effects were not found, however, for frequencies of sex-linked recessive lethals induced by mustard gas [29], EMS [134], MMS [134], or DES [134]. The mechanism by which storage of treated sperm permits detection of translocations is uncertain. It has been suggested [210] that the pretranslocation lesions require a delay to open and rejoin. The time between sperm treatment and fertilization may be inadequate for these breakage and exchange processes to occur if the sperm are not stored in the female for a period of at least 6--10 days prior to fertilization [210]. In any event, the results of Mufioz and Barnett [210] would suggest that DES is effective in inducing reciprocal translocations in Drosophila, at least under the specified treatment conditions.

Studies with other insects Studies of mutagenesis by dialkyl sulfates in insects other than Drosophila are quite limited. Murota and Murakami [212,213] have reported the induction of dominant lethal mutations by DES in the silkworm Bombyx mori. They used reduction in egg hatchability following treatment of the male parent in mid-pupal stage as an index of dominant lethality. The detection of genetic effects of DES following treatment of female pupae [319] and the activity of DES in a specific-locus test based upon egg color [211] have also been reported in abstracts. Genetic effects in fish

The induction of mutations by DMS has been reported for t w o genes affecting the morphology of scales in the carp, Cyprinus carpio [323]. Among 24 200

113 progeny produced from eggs fertilized with mutagenized sperm, two mutations were recovered at one locus and three at the other. No mutations were recovered among 260 000 control offspring. Whether such induced alterations represent point mutations or deletions is not known. The induction o f chromosomal alterations by DMS has been reported in mitotic cells of Salmo irideus, the rainbow trout [322]. Sperm treated for 2 h with 7.7 × 10 -4 M DMS were used to fertilize untreated eggs. The occurrence o f aberrant mitotic figures was scored in embryos at the blastula stage. Bridges and fragments were reported to occur at considerably higher frequencies in embryos derived from treated sperm than in the controls. Chromosomal abnormalities induced by DMS have also been reported in studies with carp sperm and embryos [324]. Genetic effects in mammalian cells in culture

Induction o f mutations and chromosome aberrations The induction by dialkyl sulfates of mutations conferring resistance to 6-thioguanine has been studied in Chinese hamster ovary cells [59]. Linear dose--response curves were reported for both DMS and DES. DMS was more cytotoxic than DES and also induced a higher mutation frequency when the chemicals were compared at equal concentrations. When the comparison was made at equal levels of survival, however, the mutation frequency induced by DES was higher than that induced by DMS [59]. Published data on the induction of chromosome aberrations in mammalian cells b y dialkyl sulfates are quite limited. DMS has been reported to induce chromatid aberrations in mouse ascites t u m o r cells [261] and, in combination with caffeine, to induce chromosome aberrations in Chinese hamster cells [309]. The results, however, are n o t extensive because in both cases caffeine, rather than DMS, was the primary subject of the investigation. Induction o f sister-chromatid exchanges and unscheduled DNA synthesis It has recently been shown that a wide variety of mutagens stimulate the occurrence of sister-chromatid exchange in mammalian cells [232]. Consequently, it has been suggested that the induction of sister-chromatid exchanges may be used for the detection of mutagens [56,232,338]. Wolff et al. [338] have demonstrated that DMS is very effective for the induction of sister-chromatid exchanges in human fibroblasts. A dose-dependent responses was reported for treatments through two rounds of DNA replication at DMS concentrations from 10 -4 M to 10 -4 M. At the highest concentration there was an average of 1.23 sister-chromatid exchanges per chromosome compared to a control level of 0.31. In fibroblasts from a xeroderma pigmentosum (XP) patient (XP12RO cells), the levels of sister-chromatid exchange induced at each concentration were higher than in normal fibroblasts [338]. The spontaneous frequencies of sister-chromatid exchange are the same in both cell lines [56]. Although the XP cells exhibited reduced levels of excision o f damage induced by ultraviolet light, 4-nitroquinoline-l-oxide, or mitomycin C, they did not exhibit any reduction in excision of damage induced by DES [56], DMS, MMS, EMS, MNNG, or ENU [338]. The interpretation of the ele-

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vated levels of sister-chromatid exchange induced by DMS and DES in XP cells relative to normal cells, therefore, is not totally straightforward [338]. Cleaver [56] has suggested that the explanation may lie in a small fraction of DNA damage which is slowly or inefficiently repaired by XP cells. Such unrepaired damage may stimulate exchanges during semiconservative replication. As in the case of sister-chromatid exchanges, enhanced sensitivity of XP cells has also been noted for mutagenicity [306]. Induction of repair replication associated with excision, detected as unscheduled DNA synthesis, has recently been employed as an indicator of DNA damage induced by mutagens [257,303]. The occurrence of repair replication in XP and normal human fibroblasts following exposure to DMS or DES has been demonstrated by Cleaver [ 56]. Genetic effects in mammals

Induction o f dominant lethals Because of the ease with which it can be conducted, the d o m i n a n t lethal test in mice has been extensively used relative to other mammalian germ-cell tests. In this test, treated males are mated, and the females are dissected in mid-pregnancy to detect embryos which died in utero [32,77]. In a study by Epstein and 8hafner [77], DMS was tested but was not shown to induce d o m i n a n t lethals in Swiss mice (CD-1). At a dose of 23 mg/kg administered by intraperitoneal injection, there was only one deciduoma out of 150 total implants scored from 14 female mice. This number is n o t significantly different from the reported control value of 12 deciduomata from 1434 total implants. The DMS dose used was roughly equivalent to an LDs dose. The data presented for DMS [77] were based upon females mated during the third week following treatment of the males. For MMS, it is noted [77] t h a t d o m i n a n t lethal effects were pronounced in the second week but declined to control values by the third week. In view of the similarities of action of DMS and MMS, a comparable effect with DMS would not be totally unexpected. It is not clear from the experimental protocol whether data were collected for matings two weeks after DMS treatment. Data are not presented, however, and the negative result for DMS is n o t discussed. Another possible explanation for the negative result with DMS is the rapid breakdown of the c o m p o u n d in whole mammals. Swann [317] has found, for example, that DMS can no longer be detected in blood 3 min after administration of a dose of 75 mg/kg to rats by injection into the tail vein. In a comparable study with MMS at 100 mg/kg, the c o m p o u n d was still detectable in the blood 1.5 h after treatment. Malashenko and Egorov [184] have reported the induction of d o m i n a n t lethals in mice by DES. When male mice were given one intrascrotal injection at 6 mg/kg, no increases in d o m i n a n t lethality were observed for matings in the first two weeks after treatment. For mating in the third and fourth weeks, however, an increase was detected, indicating induction of d o m i n a n t lethals in early spermatids and spermatocytes but not in late spermatids or spermatozoa. The frequencies in the treated groups were from 1.7 to 1.9 times those of the controls. The controls in these experiments, conducted with C3H males and CBA females, however, showed high and somewhat variable spontaneous frequencies of d o m i n a n t lethality, ranging from 9.9 to 15.5%.

115 In later experiments with C57BL/6 males and CBA females, Malashenko [182] confirmed his result for the induction of dominant lethals in spermatocytes by DES at the low dose of 6 mg/kg. In the same study, no significant increases in dominant lethality were reported at a DES dose of 30 mg/kg. A single administration of DES at 150 mg/kg, however, yielded significant increases in dominant lethality for matings corresponding to treatment of spermatids. On the basis of these results, Malashenko [182] proposed that the testis includes a population of cells that are highly sensitive to DES and that the induction of dominant lethals by low doses may be attributed to these cells. At somewhat higher doses dominant lethals are not detected because the sensitive cells are killed. At sufficiently high doses dominant lethals are induced in the more resistant cell population. Unfortunately, spontaneous frequencies that are somewhat high and variable (5.7--10.2%) again complicate interpretation of the results. At the 150 mg/kg dose, for example, an induced frequency of 13.4% for late spermatids is not significantly different from its control whereas an induced frequency of 12.2% for early spermatids is reported to be significantly different from its control. Malashenko and Egorov [184] have conducted dominant lethal tests involving subchronic, as well as acute, exposures to DES. When animals were given 6 weekly intraperitoneal injections of DES at doses of 6 mg/kg, increases in dominant lethality were observed for matings taking place between 40 and 55 days after the last treatment [184]. The results were interpreted in terms of dominant lethals induced in spermatogonia. In their totality, the data indicate the induction of dominant lethals in mice by DES. Because of high and variable control levels of dominant lethality, however, results with respect to stage-sensitivity and the existence of cell populations of differing sensitivity must be regarded as preliminary. It would, in addition, be premature to propose a fundamental difference between DES at low doses and EMS or MMS which are reported to induce dominant lethals predominantly in late spermatids and spermatozoa [53,70]. The experiments of Malashenko and his coworkers [182,184,185], nevertheless, raise interesting questions pertaining both to the effects of DES and to the nature of the dominant lethal test. For example, it has been noted [185] that there are differences among strains of male mice in susceptibility to the induction of dominant lethals by DES.

Induction of cytogenetic alterations Surkova and Malashenko [312] reported the detection of induced aneuploidy but not chromosome breakage in bone-marrow cells of CBA mice treated with DES. The doses were 150 and 200 mg/kg administered by intraperitoneal injection. Approx. 10-fold increases in the frequency of aneuploidy above the spontaneous level of about 0.35% were reported at times between 24 h and 30 days post-treatment. To look for meiotic abnormalities induced by DES, L6onard et al. [169] treated 12-week-old male BALB/c mice by intraperitoneal injection at a dose of 177 mg/kg. Meiotic preparations were made 120 days later to permit detection of chromosome rearrangements induced in spermatogonia by examining spermatocytes. Examination of 100 spermatocytes per testis and a total of 2000

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spermatocytes revealed no chromosome rearrangements. Similar negative results were reported for several other mutagens including EMS [167,168] and MMS [168]. Both these c o m p o u n d s were positive, however, in a test for translocations in the F~ offspring of treated males [167,168]. While comparable heritable translocation data are not published for DES, it would not be unexpected for DES to be positive in view of its similarities with EMS. The only c o m p o u n d s which were scored as unambigously positive in the spermatocyte test for chromosome rearrangements in treated males are the polyfunctional alkylating agents triethylenemelamine {TEM) and triethylenephosphoramide (TEPA} [167]. It would seem, therefore, that evaluating the meaningfulness of the negative result obtained with DES in this test must await further characterization and validation of the test. L~onard [168] has suggested that the basis for the difference between the spermatocyte test and the heritable translocation test may lie in the germ-cell stages involved. The positive results in the heritable translocation test may indicate the sensitivity of spermatids and spermatozoa to the mutagens. The negative results in the spermatocyte test in treated males may indicate lower susceptibility of spermatogonia to these agents. In addition to studies with experimental animals, there is one report in which chromosome aberrations were scored in l y m p h o c y t e s of chemical workers who may have had occupational exposures to DMS. DMS was n o t detected in air samples, however, and elevated levels of aberrations were not found [91]. No conclusion can be drawn, therefore, from the negative result.

Induction o f specific-locus mutations The effects of DES in a mammalian specific-locus test have been studied by Malashenko [183] who used YT females homozygous for 7 visible m u t a n t alleles and C3H males. The males were treated by intraperitoneal injection at a dose of 5 mg/kg twice weekly for 10 weeks. 5 weeks after the termination of treatment, they were mated to the YT females. 1 m u t a n t and 4 mosaic mutants were reported among 5042 F1 mice compared to none in the control. Malashenko [183] concluded that DES is mutagenic in spermatogonia in the specificlocus test. Another report of DES-induced mutations in mice is that of Egorov and Blandova [69] involving changes in histocompatibility. Males (C57BL/ 10ScSnEg, D2(H-2d)) were given weekly intraperitoneal injections of DES at individual injection doses from 6 to 200 mg/kg for 6 weeks. 60 days after the first injection, mating was begun between treated or control males and C57BL/ 10ScSnEg(H-2 b) females. Histocompatibility was tested by detecting rejection of reciprocal skin grafts made between the F1 mice at ages of 6--8 weeks and standard F I hybrids of the same genetic background. A statistically significant increase in the frequency of graft-rejection was recorded, and it was concluded that DES induced mutations in the H-locuS system. No dose dependence was observed, however, and the frequency of mutations was actually lower at total doses of 600 and 1100 mg/kg than at 36 mg/kg. Comparable increases in frequency of histoincompatibility were n o t found in the progeny of treated A/SnK1Y(H-2 a) males and A/SnK1Y,CA(H-2 ~) females. Although of considerable interest, the results of this study should probably be regarded as pre-

117 liminary with respect to mutagenesis by DES. Reasons for this interpretation include that unexplained strain difference, the apparent low efficiency of DES at the higher doses tested, and the relatively small number of total mutants [16] upon which the study is based.

Inhibition o f testicular DNA synthesis DMS and DES have been shown by Seiler [264] to inhibit testicular DNA synthesis in the mouse. It has been suggested by Friedman and Staub [88] that inhibition of testicular DNA synthesis can be used as a simple mammalian assay to detect genetically active chemicals. Mice axe given an intraperitoneal injection with tritiated thymidine 3.5 h after chemical exposure and are killed 0.5 h later to measure uptake of the labeled nucleoside into DNA [88]. It is reported that a number of mutagenic and carcinogenic substances are active in this assay, while a number of putative non-mutagens, including compounds that are highly toxic or have carcinogenic analogues, are not [88,264]. DMS, administered at a dose of 100 mg/kg by intraperitoneal injection, caused a reduction in testicular DNA synthesis to 26.2% of the control level. Similarly, after treatment with DES at a dose of 200 mg/kg given by the same route, incorporation of tritiated thymidine into DNA was 17.8% of the control value. Note added in Proof I would like to bring to the reader's attention articles by E. Vogel and A.T. Natarajan that have been published since this review went to press. The articles concern the effects of monofunctional alkylating agents, including DMS, in Drosophila. The first (Mutation Res., 62 (1979) 51--100) reports on recessive lethal mutations and translocations, and the second (Mutation Res., 62 (1979) 100--123) is concerned with total or partial sex chromosome loss.

Acknowledgements I would like to thank Mr. John Wassom and his staff at the Environmental Mutagen Information Center, Oak Ridge, TN, for providing computerized literature searches and copies of a number of articles. I also thank Dr. Robert Racine for his assistance with German translations, Mrs. E.S. Von Halle for editing the bibliography, and Drs. Michael Shelby, Daniel Straus, and Errol Zeiger for reviewing the manuscript. The secretarial and organizational work of Mrs. Linda Hoffmann is also gratefully acknowledged.

References 1 Aastveit, K,, Effects o f c o m b i n a t i o n s o f m u t a g e n s o n m u t a t i o n frequency in barley, in Mutations in Plant Breeding II0 I A E A , V i e n n a , 1 9 6 8 , pp. 5--14. 2 Abraham, S.K., V. Goswami and P.C. Kesavan, Mutagenicity o f inhaled d i e t h y l sulphate v a p o u r in Drosophila melanogo~ter and its i m p l i c a t i o n s for the utility o f the s y s t e m for screening air pollutants, Mutation Res., 66 (1979) 195--198. 3 Abrahamson, S., an d E.B. Lewis, T h e d e t e c t i o n o f m u t a t i o n s in Drosophila melanogo~ter, in: A. Hol]aender (Ed.), Chemical Mutagens, Principles and M e t h o d s for their D e t e c t i o n , Vol. 2, Plenum, N e w York, 1971. pp. 461--487.

118 4 A d l e r , B., R. B r a u n , J. S e h S n e i c h a n d H. B S h m e , R e p a i r - d e f e c t i v e m u t a n t s of P r o t e u s mirabilis as a p r e s c r e e n i n g s y s t e m f o r t h e d e t e c t i o n of p o t e n t i a l c a r c i n o g e n s , Biol. Z e n t r a l b l . , 95 ( 1 9 7 6 ) 4 6 3 - - 4 6 9 . 5 A l d e r s o n , T., E t h y l a t i o n versus m e t h y l a t i o n in m u t a t i o n o f Escherichia coli a n d D r o s o p h i l a , N a t u r e ( L o n d o n ) , 203 (1964) 1404--1405. 6 A l d e r s o n T., C h e m i c a l l y i n d u c e d d e l a y e d g e r m i n a l m u t a t i o n in D r o s o p h i l a , N a t u r e ( L o n d o n ) , 207 (1965) 164--167. 7 Alderson T., Spontaneous and induced reversion of ICR-170-induced xanthine dehydrogenase mutants of Aspergillus nidulans, Mutation Res., 8 (1969) 521--529. 8 Alderson T., and A.M. Clark, Interlocus specificity for chemical mutagens in Aspergillus nidalans, Nature (London), 210 (1966) 593--595. 9 Alderson T., and M.J. Hartley, Specificity for spontaneous and induced forward mutation at several gene loci in Aspergillus nidulans, Mutation Res., 8 (1969) 255--264. 10 Alderson T., and M. Peleeanos, The mutagenic activity of diethyl sulphate in Drosophila melanogaster, II. The sensitivity of the immature (larval) and adult testes, Mutation Res., 1 (1964) 182--192. 11 Alderson T., and C. Scazzocchio, A system for the study of interlocus specificity for both forward a n d r e v e r s e m u t a t i o n in at least e i g h t gene loci in Aspergillus nidulans, M u t a t i o n Res., 4 ( 1 9 6 7 ) 5 6 7 - 577. 12 A l i k h a n y a n , S.I., a n d G.M. N a l b a n d y a n , S e l e c t i o n o f w i n e y e a s t s using m u t a g e n s , C o m m u n a t i o n I. 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