[CANCER RESEARCH 40, 4704-4708, December 1980]0008-5472/80/0040-0000302.00

Mutagenicity of Hydroxamic Acids and the Probable Involvement ofCarbamoylation1

Paul L. Skipper,2 Steven R. Tannenbaum, William G. Thilly, Elizabeth E. Furth, and Walter W. Bishop

Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

ABSTRACT

A series of hydroxamic acids (aceto-, propiono-, benzo-, andp-nitrobenzo-) and seven derivatives of these were examined

for biological activity using Salmonella typhimurium. Acylationto yield O-acetyl and O-benzoyl derivatives markedly enhanced

toxic properties and was necessary for mutagenic activity forall but p-nitrobenzohydroxamic acid. The dose necessary to

produce a minimum significant mutagenic response varied from21 IJLMfor O-benzoyl benzohydroxamate to 430 fiM for O-acetyl

acetohydroxamate. These two compounds were also testedwith human lymphoblasts to which they were toxic at 100 /¿Mbut not mutagenic. O-Acetyl benzohydroxamate, a mutagen,was prepared with a 14C label in the carbonyl carbon atom of

the benzoyl group and was shown to form an adduct in vitrowith DMA and polyguanylic acid. The level of binding was 1mol of 14Cper 5x10" mol of DMA phosphate and 1 mol of 14Cper 105 mol of polyguanylic acid phosphate.

INTRODUCTION

A large number of chemical mutagens and carcinogens aredirect-acting alkylating agents (9). A/-alkyl-A/-nitrosoureas are

potent members of this class of compounds and thus havereceived extensive investigation into the chemicobiological interactions through which their clinical effects become manifest(11). These investigations have focused largely on the alkylating nature of the nitrosoureas. Other modes of reactivity arealso present, however, and the interpretation of experimentalresults has not successfully been based solely on alkylatingactivity.

Alkyl nitrosoureas [R-N(NO)—CONH-R'] decompose inaqueous solutions forming at least 2 short-lived intermediates,isocyanates (R'—N=C=O) and diazotate ions (R—N=N—O")

(13). The latter, by protonation and subsequent loss of hydroxide, yield diazonium ions (R—N+=N), which are powerful al

kylating agents. The isocyanates react with available nucleo-philes (X), adding a carbamoyl group (X—CONH—R').

Carbamoylation measurement has been reported for W-chlo-roethyl-A/'-cyclohexyl-A/-nitrosourea which carried a 14C label

in the cyclohexyl group (2). No Carbamoylation of purified DNAor synthetic polynucleotides in vitro was detected, and thepossibility was raised that the low-level cellular DNA binding

may have arisen from reaction with nuclear proteins tightlybound to isolated DNA. Carbamoylation of proteins by nitrosoalkylureas has been documented (1, 2, 16), and the e-amino

group of lysine (15) and AMerminal amino groups (26) havebeen shown to be the principal sites of reaction.

1This work was supported by Grant 2-P01-ES00597-09 from the National

Institute of Environmental Health Sciences. USPHS.2 To whom requests for reprints should be addressed.

Received May 2. 1980; accepted September 2, 1980.

In our present study, we examine the possibility that carba-

moylation alone may create premutagenic lesions in DNA. Thecarbamoylating reagents used are O-acyl hydroxamic acids,

which are moderately stable compounds that readily rearrangeto isocyanates (27). Thus, we avoid the complications of concomitant alkylation inherent in the use of alkylnitrosoureas ascarbamoylating agents as well as the extreme reactivity associated with isocyanates.

MATERIALS AND METHODS

Mutation Assays. Forward mutation to 8-azaguanine resist

ance in Salmonella typhimurium strain TM 677 (uvrB, rfa, pKM101, gal', bio', his*) served as the principal means of meas

uring mutagenicity. Reversion to histidine prototrophy wasquantitatively measured for 2 O-acyl hydroxamates using S.

typhimurium tester strains TA 1535, TA 1537, TA 1538, TA98, and TA 100 kindly supplied by Professor Bruce Ames,University of California at Berkeley. Specific protocols for quantitative measurement of forward and reverse mutation havebeen described in detail previously (18, 19). Briefly, exponentially growing bacteria were suspended in medium in the presence of the test chemical for 2 hr and then resuspended infresh medium. Aliquots were plated in the absence and presence of selective conditions (medium containing 50 ¿ig8-azaguanine per ml or histidine-deficient medium with agar). Two

independent cultures were used for each treatment point.Colonies were counted at 48 hr, and the mutant fraction wasdetermined as the number of colonies formed under selectiveconditions divided by the number of colonies formed undernonselective conditions multiplied by the dilution factor. If thisratio for a treated culture was greater than that found foruntreated cultures used as simultaneous controls with greaterthan 99% confidence and if that ratio also exceeded the 99%upper confidence limit on the mutant fractions of all historicalcontrols, the test is considered positive.

The historical background mutant fractions and 99% upperconfidence limits for the bacterial strains are: TA 1535, 3.1and 7.4; TA 1537, 6.8 and 19.4; TA 1538, 2.4 and 8.8; TA 98,4.0 and 11.8; TA 100, 22 and 57; TM 677, 7.5 and 17. ForTM 677, these data are x 105, and for the others, the data arex 10s. The TM 677 data are based on the more than 400

experiments performed in our laboratories to date. The valuesare calculated as described in Ref. 18, which is also the sourcefor the other values.

Human Lymphoblast Mutation Assay. Diploid human lymphoblasts, line TK 6, were used to determine if O-benzoylbenzohydroxamate or O-acetyl acetohydroxamate were mutagenic to human cells. The protocol consisted of 24-hr exposure

in Roswell Park Memorial Institute Medium 1640 supplementedto 15% with fetal calf serum. The culture was sampled at theend of the treatment period, and cell survival was determined

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Mutagenic Hydroxamic Acids

by clone formation in microtiter plates. Cultures were maintained in exponential growth for an additional 6 to 8 days topermit phenotypic expression of any induced mutation, andsamples were then plated in the absence or presence ofselective conditions (medium containing 1 ¿igtrifluorothymidineper ml or 5 fig 6-thioguanine per ml). Colonies were enumerated

after 14 days, and the mutant fraction was calculated as theratio of colony-forming efficiency in the presence and absence

of selective conditions. The assay process is discussed indetail elsewhere (21).

Chemicals. Purified DNA from calf thymus (type 1) waspurchased from Sigma Chemical Co., St. Louis, Mo. Polyguan-ylic acid was purchased from Collaborative Research, Wal-

tham, Mass. Acetohydroxamic acid was obtained from AldrichChemical Co., Milwaukee, Wis.

Propionohydroxamic acid (8), benzohydroxamic acid (6), p-nitrobenzohydroxamic acid (4), and N-methylbenzohydroxamic

acid (14) were prepared according to published procedures aswere the benzoyl esters of benzohydroxamic acid (10) andacetohydroxamic acid (7).

The acetyl esters were prepared according to the followingprocedure. The hydroxamic acid was dissolved in tetrahydro-

furan to which were added, with stirring, 1.1 equivalents ofpyridine followed by 1.1 equivalents of acetic anhydride. Themixture was held at room temperature until esterification wascomplete as determined by the absence of color reaction withFeCI3. The solvent was evaporated, and the residue was takenup in ether which was washed twice with 1 N HCI. The productwas obtained by evaporation of ether after drying. With theexception of O-acetyl W-methylbenzohydroxamate which wasan oil, all the products were known compounds and had correctmelting points. All hydroxamic acids gave an intense red coloration with FeCI3, and all O-acylated hydroxamic acids did

not.O-Acetyl [7-14C]benzohydroxamate was synthesized accord

ing to the following procedure. Benzoic acid (2.9 mg; 0.0238mmol; 0.5 mCi), purchased from New England Nuclear, Boston,Mass., was mixed with benzoyl chloride (141 mg; 1 mmol) andheated at 100°for 1 hr. The resultant mixture was dissolved inether (2.5 ml) and ac'ded to a mixture of Na2CO3 (106 mg; 1

mmol) and NH2OH-HCI (70 mg; 1 mmol). Water (175 /il) wasadded, and the mixture was stirred 40 min. The ether solutionwas removed, and the residue was extracted with ether until allthe product was separated from the aqueous phase. Thecombined extracts (~13 ml) were evaporated to dryness, and

the crystals that remained were recrystallized twice from ether.Thin-layer chromatography on silica gel using toluene:dioxane:acetic acid (20:5:1) revealed the product to have 97% radio-purity. Yield was 96 mg (70%). The entire product was treatedwith acetic anhydride and pyridine as described above to yield91 mg of colorless crystals (0.51 mmol; 50%). Radiopurity bythin-layer chromatography (ether) was 95%. The final product

had a specific activity of 0.47 mCi/mmol.Binding Studies. DNA or polyguanylic acid (5 mg) was

dissolved in 0.05 M Tris buffer (1 ml) to which was added O-acetyl [7-'4C]benzohydroxamate (3.8 mg; 10 ¿iCi).The mixture

was held overnight at ambient temperature followed by theaddition of 2 volumes of ethanol to precipitate macromolecules.The supernatant was removed, and the precipitate was redis-

solved in 0.05 M Tris buffer (2 ml) which was applied to acolumn of Bio-Gel P-6 (1.5 cm x 60 cm). The column was

eluted with the same buffer at a rate of 10 ml/hr. The absorb-

ance of the eluant at 260 nm was monitored continuously.Fractions (2 ml) were collected and counted in 10 ml Aquasolwith a Beckman scintillation counter.

RESULTS

Bacterial Mutation. Eleven substituted and free hydroxamicacids were used in forward mutation studies in concentrationsranging from 8 /XMto 13.3 mw. Table 1 lists the compoundswith their structures and gives the mutant fractions and survivals observed from each concentration tested. Confidencelimits (99%) are included for each mutant fraction in the table.Of the compounds tested, 4 were not mutagenic in the concentration range tested: acetohydroxamic acid; propionohy-droxamic acid; benzohydroxamic acid; and O-acetyl /v-meth-

ylbenzohydroxamate. All but acetohydroxamic acid displayedsome toxicity at high concentrations. The other 7 compounds(O-acetyl acetohydroxamate, O-benzoyl acetohydroxamate, O-acetyl propionohydroxamate, O-acetyl benzohydroxamate, O-benzoyl benzohydroxamate, p-nitrobenzohydroxamic acid,and O-acetyl p-nitrobenzohydroxamate) were found to be both

toxic and mutagenic. No compounds displayed mutagenicity inthe absence of significant toxicity.

A common and possibly interesting feature of the dependence of mutant fraction on concentration was the apparentreduction of mutant fraction when survival had been lowered to10 to 30% of untreated controls. Such a reduction implies thatthe assumption of independence of cell killing and mutationmay not be valid at the higher concentrations tested or thatsome unsuspected biological phenomenon such as the existence of a period of cell growth cycle refractory to treatment isoperative. This phenomenon is illustrated in Chart 1 in whichthe observations for O-benzoyl acetohydroxamate and O-ace

tyl benzohydroxamate are presented as representative findings. Also common was the finding of significant mutagenicityat concentrations of 50 to 100 fiw, which ranks this compoundclass among the more potent of chemical mutagens. For instance, in other experiments performed in our laboratories, wehave determined that methyl methanesulfonate and methylnitrosourethan induce significant mutation at concentrations of70 and 5 /xM, respectively.

Table 2 summarizes results obtained with O-acetyl acetohydroxamate and O-benzoyl benzohydroxamate in several reversion assays. The numbers of mutants produced in these assaysby these 2 compounds were for the most part quite small;consequently, the 99% confidence limits are quite large. Bothcompounds have elevated mutant fractions relative to the historical controls for all strains, with one exception: O-benzoyl

benzohydroxamate and TA 1535. However, 5 combinationsare not significant with 99% confidence. These are O-acetylacetohydroxamate and TA 1535, TA 1537, or TA 1538 and O-

benzoyl benzohydroxamate and TA 1535 or TA 1538.Human Lymphoblasts. Chart 2 summarizes the observations

of toxicity and mutagenicity with TK 6 line diploid humanlymphoblasts. Cells tolerated 24-hr exposures to both O-benzoyl benzohydroxamate and O-acetyl acetohydroxamate up to10"6 M, but 10~" M was toxic to 35% of the cells in the former

and 85% of the cells in the latter case. For neither compoundat either gene locus tested was a mutagenic response significant at the 99% confidence limit observed, the highest con-

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P. L. Skipper et al.

TableMutagenicity

of hydroxamic acidsintyphimuriumCompound

andstructureAcetohydroxamic

acido^^NH-OHO-Acetyl

acetohydroxamateJL

JL^^NH-O^^O-Benzoyl

acetohydroxamateMM

O^QPropionohydroxamic

acidqO-Acetyl

propionohydroxamatevAiH-ABenzohydroxamic

acidOf^V^NH-OH(Ljj^^O-Acetyl

benzohydroxamatef?

Mj^y^NH-O^^O-Benzoyl

benzohydroxamateo

or^^tr^'^^^NH—Q^^^r^''^^y^ji

k/O-Acetyl-N-methylbenzohydroxamateo

or^V^N-cr^^^

¿Hp-Nitrobenzohydroxamic

acid

Piforward

mutation assay usingS.TM677Con

centration(flM)1,33013.300170430pfinÖDU2.5604.2708,55056

1102801

.12056110220

5601,1203876150

3807607309101

,2201,8203.6507.300561102801,1208214183521

nn1UU2601.0405,240110550Relativesurvival1.01.00.70.5n

AU.40.50.40.40.6

0.30.20.11.01.01.0

1.01.01.00.90.8

0.60.31.00.91

.00.90.70.40.6

0.40.20.21.00.70.40.20.9[\

QU.tl0.90.70.21.0

0.5Mutant

fraction(xl05)a2.5

±0.34.1±0.411

±222±4Q-7_i_O•J

I XO20

±424±618

±537

± 7103 ±22

33 ±1321

±1115

±322

±520± 4

8.4 ±217±318

±340

±722

± 526 ±660

±2111

±310

±311±3

14 ±47.4±34.6±328

± 548 ±951

±1427±89.5

±319±542±831

±117.6

±2'' ' Ì *-Oo.y ± ¿.

6.8 ±26.0±27.2±35.7

± 0.729 ± 4IJU

II20I

IO100

t (°)(Wo90|\S3801\270\£

60 1\i

90 1 \ L—T--^.«4O 1 1 *~~~~~"~""---~^530 I t——__ T ¡[ ' ""'"'—~~-J"

' TT20'•IOi

n§

1.0Å“

°'8(/>0.6 • n

V 04 \ •i 0.2 K.^._i

• *•0

0.5 1.0 0 0.5![DOSE

mM]Chart

1. Mutant fraction and survival as a function of dose in theforwardassay(TM 677) for O-benzoyl acetohydroxamate (a) and O-acetyl benzohydrox

amate (£>).The dashed line represents a minimum value for the mutantfractionabovewhich the response is considered positive with 99% confidence. Theerrorbars

are 99% confidence limits on eachpoint.Table

2Mutagenicity

and toxicity of hydroxamic acids in several reversionassaysCon

centration Relative MutantfractionCompound

Strain (/IM) survival (xlu")O-Acetyl

acetohydroxa- TA 1535 860 0.3 15 ±11amateTA 1537 860 0.4 22 ± 19aTA

1538 860 0.4 14 ±11aTA98 430 0.5 24 ±16a860

0.1 72 ± 566

TA 100 170 0.7 88 ±264300.6 190 ±42b8600.4 390 ±93bO-Benzoyl

benzohydrox- TA 1535 41 0.6 2.9 ±12aamate82 0.3 5.2 ±21sTA

1537 41 0.5 63 ±316820.1 157±117bTA

1538 41 0.5 16 ±10aTA98 41 0.5 42 ±32fcTA100 41 0.8 150 ±36°82

0.2 506 ±1806a

Not greater than historical control with 99%confidence.faGreater than historical control with 99%confidence.Binding

to DNA and Polyguanylic Acid. Reaction of O-acetyl[7-14C]benzohydroxamate with DNA yielded 2 x 10~5 mol of

O-Acetylp-nitrobenzohydroxamateo<^^^*sjcyOjN-^i^OIH

er^2245892204500.90.70.60.40.31215265888±

3±3±

5±11±

18

Values greater than 17 are indicative of mutagenicity with greater than 99%confidence.

centration tested being 10 4 M. Both negative and positive

controls (ICR-191; 1 ¿ig/ml)produced mutant fractions withinthe range of historical controls. Methyl nitrosourea by way ofcomparison is significantly mutagenic to both loci at a concentration of 1CT5 M (22).

14Cbound per mol of DNA phosphate and 1 x 10 5 mol of 1*C

bound per mol of polyguanylic acid phosphate. These valuesare calculated from the weight of DNA or polyguanylic acidapplied to the column and the total radioactivity present in thefractions of column effluent-containing material excluded bythe column and absorbing at 260 nm. Correspondence ofradioactivity with absorbance was exact. Specifically, 180 and85 dpm were observed for 5 mg DNA and polyguanylic acid,respectively.

DISCUSSION

Carcinogenic, mutagenic, and cytotoxic activity have beenobserved previously for hydroxamic acids (3, 5, 23, 24). Perhaps the best characterized of these are derivatives of N-

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Mutagenic Hydroxamic Acids

05

-A»—A»—A»,

BBHS

A6TGRAF3TdRR

AAAH•Oa

-A»

AO

O

io

<<

-6 -5 -3LOG|0 CONCENTRATION(M )

Chart 2. Cell survival (Si and mutation to 6-thioguanine resistance (670") ortrifluorothymidine resistance (F3TdR") in diploid human lymphoblasts exposed to

acetyl acetohydroxamate (AAH) or benzoyl benzohydroxamate (BBH). Initialconcentrations of a 24-hr exposure are given.

hydroxy-A/-acetyl-2-aminofluorene. These compounds have an

electrophilic amino group which reacts with nucleophilic sitesof DMA. The W-hydroxy group, which is usually esterified, acts

as a leaving group in a nucleophilic substitution reaction whichmay be bi- or unimolecular (17). A necessary structural feature

of these carcinogens is the bond between an aromatic moiety(fluorene) and the nitrogen atom, which permits delocalizationof the electron deficiency generated at the amino nitrogen inthe transition state of the reaction with DNA. Such delocalization may even lead to substitution at sites distant from butconjugated with the amino nitrogen (25). The hydroxamic acidswe have studied bear a hydrogen atom in place of an aromaticnucleus; consequently, we expect the mechanism of action tobe fundamentally different.

We propose that the hydroxamic acids which are not substituted on the nitrogen atom react by a mechanism involvingrearrangement to isocyanates as electrophilic intermediates.Such a rearrangement is known as the Lossen rearrangementand is typical of hydroxamic acids and their derivatives whichbear no substituent on the nitrogen atom. It was first observedby Lossen (10) in 1872 when he heated O-benzoyl benzohy

droxamate above its melting point and observed the formationof phenyl isocyanate. It has since been studied extensively andis well characterized (27). Of particular interest to the presentstudy is that, while virtually all hydroxamic acids, if not A/substituted, undergo rearrangement under sufficiently vigorousconditions, many O-acyl derivatives are sufficiently reactive to

form isocyanates in neutral or mildly basic solution (12, 20).Other reaction pathways which are possible alternatives to

the Lossen rearrangement include nucleophilic attack on theamine nitrogen as was discussed earlier and acylation bytransfer of the O-acyl group. Our results lead us to suggest

that such mechanisms are at least of lesser importance than

carbamoylation. For instance, O-acetyl A/-methylbenzohydrox-amate, which differs from the mutagenic O-acetyl benzohydroxamate only in the methyl substitution, was completelywithout mutagenic activity and first showed toxicity only at 5HIM concentration, whereas mutagenic hydroxamates weremeasurably toxic in 0.1 rriM concentration. Nucleophilic attackon the amine nitrogen should be facile with the /V-methyl

derivative, and the lack of bioactivity implies that it does notoccur. On the other hand, the methyl substituent does makeLossen rearrangement impossible.

Acylation by transfer of the O-acyl group is also unlikely asa result of the nonmutagenic character of O-acetyl /v-methyl-

benzohydroxamate. The methyl group, being sterically smalland distant from the acyl group, should have little influence onreactivity at that site. Further support for the idea that acyltransfer is unimportant comes from the observation that p-

nitrobenzohydroxamic acid is a mutagen in our tests althoughit carries no O-acyl substituent. The nitro group itself is unlikelyto be the source of mutagenic activity since p-nitrobenzamide

was nonmutagenic in our tests. Others have also reported thatsome hydroxamic acids, without any O-substitution, are muta

genic (5, 23, 24).The observation that radioactivity from a hydroxamic acid

can become bound to DNA in vitro suggests that in vivo bindingto DNA is responsible for the observed changes in genotypewhen bacteria are treated with hydroxamates. The adducishould include the carbonyl carbon atom of the hydroxamicacid, since this is the location of the label in the O-acetyl

benzohydroxamate which we used in our binding studies.Considering this and the known reactivity of the compoundsexamined, one may expect the adduct to consist of a carbamoylgroup bound to an as yet undetermined part of the DNA.

It is interesting to note that human lymphoblasts were mutated by neither O-acetyl acetohydroxamate nor O-benzoylbenzohydroxamate at concentrations up to 100 ¿IM.O-Acetyl

acetohydroxamate was mutagenic to S. typhimurium only atconcentrations above 200 /XM(TA 100; reverse) or 400 pM (TM677; forward), but the low survival of human cells at suchconcentrations makes these assays impractical. O-Benzoyl

benzohydroxamate was not significantly mutagenic at either of2 gene loci in human cells at 100 ¿IMbut was mutagenic to S.typhimurium at about 20 /JM (TM 677) or 40 /¿M(TA 100 andTA 1537).

It is, of course, possible that, at slightly higher concentrationsthan those tested, O-benzoyl benzohydroxamate would bemutagenic to human cells. Certainly, a 5-fold variation in sen

sitivity between bacteria and human cells would not be unexpected. Studies specifically designed to account for very lowsurvivals would be required to explore this point; further speculation does not seem justified. We must be satisfied with theobservation that neither hydroxamic acid tested was as mutagenic to human cells as is methyl nitrosourea, which possessesboth carbamoylating and alkylating activity and which is significantly mutagenic to human cells at approximately 10 p.M(22).

ACKNOWLEDGMENTS

We thank Dr. James Miller and Dr. Elizabeth Miller for helpful suggestions anddiscussion.

REFERENCES

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