- Email: [email protected]
Hydroxylamine-O-sulfonic acid: In vitro and possible in vivo reaction with DNA
Chem.-B&l. Interacrions, 7 11913) 19!%?04 @ Ekevier Scientific Publishing Company, Am.ste&m--Printed
HYDROXYLAMINE-0-SULFONK IN V1VO REACTION WITH DNA
in The Netherlands
195
ACID: IN VITRO AND POSSIBLE
HERBERT S. ROSENKRANZ Department of Microbiology, College of Pitysicians and Surgeons, Columbia University, New York, N. Y. 10032 (U.S. A.) (Received March 9th, 1973) (Revision received June Filth, 1973) (Accepted June l&h, 1973)
SUMMARY
Exposure of DNA solutions to low levels (2 - 10’V3M)of hydroxylaminc-Osulfonic acid (HOS) resulted in limited degradation accompanied by incwases in the buoyant density of the DNA. The thermal helix-coil profile of ahe DNA was not changed significantly fo!lowing exposure to HOS. Upon thermal denaturation, the treated DNA specimens exhibited buoyant density values similar to those of heated control DNA. This is taken to mean that an added function or a modified DNA base is removed upon heating. Exposure of DNA to elevated levels of HOS (,z= 0.2 M) resulted in extensive degradation which was accompanied by spectral changes: a hypercluomic shift and an increase in the wavelength of maximum absorbance. Exposure of individual deoxynucleosides to HOS also resulted in spectral changes and in the detection of new reaction products by paper chromatographic means. HOS preferentially inhibited the growth of a bacterial strain deficient in DNA polymerase. This is a property also exhibited by known mutagens and carcinogens. This is taken to mean that HOS is capable of rescting with the DNA of living cdis as well.
INTRODUCI’ION
The carcinogenicity of aromatic arnines and amides depends upon their metabolic transform? tion to the corresponding N-hydroxy derivatives’ -3. More recently it was shown that a further N-hydroxy esterification (e.g. to the corresponding sulfate) yieided the ultimate carcinogen 4-0. The present report deals with some biol Abbreviation:HOZ, hydroxylamine-O-sulfonic acid.
196
H. S. ROsENKRANZ
in vitro effects of hydroxylamine-0-sulfonic
acid (H,N-0-SO,H; MOS). This study was undertaken to determine whether this compound could serve as a model for understanding the chemical basis of carcinogenesis by aromatic amides. METHODS
Physicochemical analysis
Sedimentation coefficients were determined in a Spinco Model E analytical ultracentrifuge equipped with an uitraviolet optical system. Solutions of DNA (0.003 % in 0.15 M NaCl containing G.015 M sodium citrate) were spun at 50 740 rev./min and pictures were taken at 2-min intervals. The photographs were traced with a Joyce-Loebl Mark III B microdensitometer and sedimentation coefficients were calculated by a modification’ of the procedure of SCHACHMAN”. The banding properties of DNA in gradients of cesium chloride were determined as described by SCHILDKRAUTet al.“. Portions of the DNA together with a reference sample (Micrococcus lysodeikticus DNA, 1.731 g/cm’ for undenatured DNA and Clostridium perfringens DNA, 1.694 g/cm3 for thermally denatured DNA) were placed in a cesium chloride solution (density, 1.70 g/ml) and centrifuged at 44 770 rev./min for 24 h. The bands formed by the specimens at their equilibrium positions were photographed and traced as above. Thermal transition profiles were measured with a Beckman DU spectrophotometer according to the manner described by MARMUR AND DoTY’~. The DNA solutions were made up in 0.015 M NaCl containing 0.0015
M sodium citrate.
The midpoint (7’,,,) of the thermal helix-coil transition curve was used to characterize the specimens. Thermal denaturation was carried out by heating samples in 0.15 M NaCl containing 0.015 M sodium citrate at l??’ for 10 min and immersing them in an ice bath. Spectra of DNA and deoxynuclecside solutions were taken with a Cary Model 11 recording spectrophotometer, the absorbance at selected wavelengths was checked with a Beckman DU-2 spectrophotometer. Corresponding pairs of specimens were diluted to the same extent for direct comparison.
Microbial assay for potential carcinogens and mutagens Portions (0.1 ml) of overnight cultures of Escherichia coli W3110 (pal A+) and its DNA polymerase-deficient derivative E. coli ~3478 (pol A;) (ref. 13) in
medium HA supplemented with 5 klg thymine per m114g15,were spread onto the surface of petri plates containing 25 ml of 1.5 “/, agar dissolved in the same medium. When the surface of the agar had dried, paper discs (6.35 mm in diameter) impregnated with the substance to be tested were deposited at the center of the plates. The plates were incubated at 37” in the dark for 14 h whereupon the diameters of the zones of inhibition were determined. Each substance was tested in replicate on several occasions. A detailed description of the procedure cau be found elsewhcrei6.
EFFECT OF HOS
ON
DNA
197
RESULTS
Reaction between DNA and HOS Exposure of DNA solutions to HOS led to a limited degradation of the polydeoxynucleotide (Table I). This depolymerization was dependent upon the temperature of incubation (Table I). It is interesting that upon thermal separation of the DNA strands the sedimentation coefficients of HOS-treated DNA did not drop precipitously (Table I); this indicates that there were not many additional “hidden” singlestrand breaks. (On the other hand, DNA exposed to hydroxyurea exhibited greatly diminished sedimentation coefficients upon thermal denaturationl’, thereby indicating the presencp of many “hidden” single-strand breaks stabilized by the doublestranded structure.) Even though the sedimentation coefficients of treated DNA decreased, this wzs not accompanied by drastic changes in the thermal helix-coil transition behavior of the treated DNA (Table I). It was, however, paralleled by significant and reproducible increases in the buoyant densities of treated DNA (Table I and Fig. I). Since it has already been shown that HOS-treated DNA was not denatured (i.e. the T, was essentially unchanged), it must be concluded that the increase in buoyant density refiected chemical modification of the DNA exposed to HOS. An examination of the tracings of the DNA bands at equilibrium (Fig. i), reveals a gradual broadening of the width of the bands. This can be taken to reflect decreased molecular weights” and it supports the sedimentation velocity data (Table I). Upon thermal denaturation the control DNA exhibited the increase in buoyant density (Table 1) associated with strand separationlg. It is however very interesting that following thermal ienaturation HOS-treated DNA’s exhibited buoyant densities nearly identical to that of the heated control DNA (Table I). Since prior to heating there was a maximal difference in buoyant densities of 0.005 g/cm” between treated and untreated DNA, it would have been expected that this difference would have been maintained or even been magnified following thermal denaturation. Because this expectation was not fulfilled it may be hypothesized either that the functional group that had been attached to the DNA was heat-labile or that heating TABLE I EFFECT OF HYDROXYLAMINE-0-SULFON~C ACID ON THE PHYSICAL CHEMICAL PROPERTlESPF DNA
Calf thymus DNA t-1 mg per ml of 0.1 M phosphate buffer, pH 7.4) was incubated with 0.0019 M HOS at the temperatures indicated for 19 h, whereupon the DNA specimens were chilled and precipitated with ethanol. The DNA fibers were washed extensively with ethanol and redissolved in 0.015 M NaCl containing 0.015 M sodium citrate. -~ . ..- -- .---.----________ .I.__-..__..___.. _---- - __.__._~_. -_-_Sedimentation Coefficien Is ( S) --..-~~_----__ Before After denaturation denaturation p__.____p ____ ___
Temperature of incubation (“C) ---.--.-
Control 4 23 37
17.1 16.3 12.2 9.3
15.0 12.6 8.3 8.0
Buoyant Densit,‘es (g/cm3) ._. _.-._-. ~-After Before denaturation denaturation ~----- -
1.704 1.704 1.706 1.709
I .722 1.723 1.721 1.722
PC)
69.6 69.8 68.8 68.1
H. S. RCX3ENKRANZ
Fig. 1. Buoyant densities of normal and HOS-treated DNA. The preparation of the specimens is described in the legend of Table I. A, Control DNA; B, C and D, .DNA exposed to 0.0019 M HOS and incubated for 19 h at 4, 23 and 37”, respectively. The band at the extreme left represent the position of the marker DNA (~~cruc~~s ~~so~e~~~cusDNA, 1.731 g/cmS). Fig. 2. Spectrum of DNA after exposure to HOS. Calf thymus DNA (I mg per ml of 0.1 Mphosphate buffer, pH 7.4) was exposed to 0.64 M HOS at 23’ for 19 h. 1, Control DNA; 2, HOS-treated DNA.
eliminated an HOS-mo~fied base from the DNA. (Thus upon thermal denaturation of alkylated DNA, 7-alkylguanine residues are split-off)**. Exposure of DNA to elevated levels of HOS (i.e. 0.64 M) resulted in degradation of the’ polydeoxynucleotide to non-sedimentable material (5.1 * IO6 g x min). owever, the spectrum of such DNA specimens was altered (Fig. 2); in addition to a hyper~hromic shift-possibly reflecting DNA denaturation2’ - there was also a shift in absorption maximum from 257.5 to 265 nm.
The effects of exposure to HOS on the absorption spectra of deoxynucleosides was inv~tigated. A comparison of the spectra of thymidine and HOS-treated thymidine (Figs. 3 and 4 and Table 11) revealed a slight decrease in absorbance of the treated material. The most drastic effect was seen at pH 13. While untreated thymidine exhibited (Fig. 3) the alkali-induced hypochromic effect and the increase in Amin previously described2 I, the HOS-treated material showed almost no effect of alkali upon the spectrum except for the ap~ara~ce of a new maximum at 216 nm (Fig. 4). Upon exposure to HOS, deoxyadenosine exhibited a hypochromic shift as well as an increase in I lllLIX at pH 7 (Fig. 5). This was paralleled by similar shifts in acid and alkali (Table la).
DNA
199
Fig. 3. Spectra of unmodified respectively.
thymidine. Curves 1, 2 and 3 represent spectra at pH 2, 7 and 13,
EFFFKT OF Has
ON
Fig 4. Spectra of HOS-modified thymidine. Thymidine (1 mg per ml of O.! M phosphate buffer, pH 7.4)was exposed to 0.24 M HOS at 54” for 90 h. The spectra of the treated material were read against blanks containing HOS. Curves I,2 and 3 represent spectra at pH 2, 7 and 13, respectively.
TABLE II SPECTRAL PROPERTIES OF DEOXYNUCLEOSIDES
HOS-treated _~_I___
Compound
il rnRZ
Lin
Deoxyadenosine
2 7 13
256 258 260
230 226 229
262 2162 261
230 230 226
Deoxyguanosine
2 7 13
253 250 266
235 223 231
248 248 245
224 225 223
Deoxycytidine
2 7 13
278 271 270
240 246 246
278 278 269
240 240 250
Thymidine
2 7 13
267 266 266
235 235 245
266 266 266 _---
235 235 236
Deoxyguanosine also showed a decreased absorbance upon exposure to HQS (compare Figs. 6 and 7). However, the most pronounced changes were decreased A,,, and &in values especially in alkali and the appearance of a new a.bsorption peak at 216 nm (Fig. 7 and Table II). Upon exposure to HOS the spectrum at neutral pH of deoxycytidine was altered drastically (Fig. 8). There was an increase in absorbance as well as an increase
Fig. 5. Spectrum of HOS-modified deoxyadenosine. The conditions of the reaction are described in the legend of Fig. 4. A, ~~~adc~~ine; B, HOS-modified d~~adenosine; pH 7.0. Fig. 6. Spectra of unmodified deoxyguanosine. Curves 1,2 and 3 repmsent spectra at pH 2,7 and 13, respectively.
Fig. 7. Spectra af HOS=modifIed daoxyguanosinc. Tttc conditions of incubation arc givon in the legend of Fig 4. Curves 1, 2 and 3 reprcsent spectra at pH 2, 7 and 13, respectively; pH 7.0. Fig, R. Spectrum of HOS-mad&d deoxycytidine. The conditians of incubation were similar to those described in the legend of trig. 4. 1, Deuxycytidine; 2, HOS-modified dcoxycytidino, pH 7,O.
in A,,,,,and Amln, Iln acid the spectra of the two preparations were quite similar while in foci they had diffe~nt Rmtn values (Figs, 9 and 10 and Table II). These spectral changes were paralleled by the appearanrx of new, as yet uni~entifi~, reaction products detectable by paper chromatography (Table III).
EFFECTOF HOS
ON DNA
Fig. 9. Spectra of unmodified deoxycytidine. Curves I, 2 and 3 represent spectra at pH 2,7 and 13, respectively. Fig. 10. Spectra of HGS-modified deoxycytidine as a function of pH. Curves 1, 2 and 3 represent spectra at oH 2, 7 and 13, respectively.
TABLE III PAPER C~R~~~~P~C
MIGRATZDt4 OF DEO~UC~~ID~
AND REACTION PRODUCTS
Solvent system: n_Butanol-Water (86: 14 v/v). Rate of m&ration
&ox_vnuciei.&ies ~~-.-Dcoxyadenosine ~oxy~~~ine Thymidine Deoxycytidine
-
Confrol
Reacted with HOS
0.31 0.15 0.49 0.20
0.41 0.37 0.56 0.39
-
_--_l__
CelIs exposed ta agents which a&et the cellular DNA attempt to repair damage
to their DNA by various repair mechanisms. One of the enzymes rvrvolved in DNA repair is DNA polymerase I (refs. 13, 22). Cells deficient in this enzyme (pal A;) have been found to be more sensitive than their pal Ai parents to the inhibitory action On the other hand, the two strains will of agents which affect cellular DNA L3*16*23. be equally sensitive to agents which affect the structure and function of other cellular components’6. Advantag: was taken of this preferential sensitivity of pot A; cells towards agents which alter cefhdar DNA to devise a simple microbian bioassay for such agents’” (see also ref. 24). In the present study it was shown that HOS inhibited the pol A; strain prcferentially (Table IV), a property it shares with the carcinogens methyl methanesulfonate and N-nitroso-IV-methylurethane, and with the mutagen mitomycin C. These agents are known to react with DNA.
H. S. ROSENKRANZ
202
On the other hand, substances known to affect cellular structures other than DNA, (e.g. methicillin, streptomycin and chloramphenicol) affected the two strains similarly (Table IV). TABLE IV EFFECT OF AGENlS ON ‘THE GROWTH OF The
DNA
POWbU3tA!&DEFlCIENT
CELLS
procedure described in the text was followed. Substances l-3 served as “positive” controls;
ia. they inhibited the pol AI- strain preferentially. Their mode of action is known to involve alteration of the cellular DNA. Substances 4-6 served as “negative” controls; they are known to affect structures other than the cellular DNA. .-
No. Agent
Amount
Diameter of zone of inhibition (mm) -..
Methyl methanelulfonate N-Nitroso-IV-methylurethane Mitomycin C Methicillin Streptomycin Chloramphenicc I HOS ______.___ .._. _-_.
-.-_.
0.13 0.02 1 30 10 30 0.14 _
ftmoles jcmoles /% Pg I% PI3 /“moles -...--__.-- --_.-__
Pol A+
Pal AI._ ___. __._
54.8 18.0 37 28.8
42.0 0 23 28.8
17
17
34 43.4
.-.
34 53.8 .._.-___.
_
DISCUSSION
Upon exposure to HOS, DNA is degraded. This depolymerization appears to result from a reaction of HOS with DNA bases as evidenced by the increased buoyant density of HOS-treated DNA. Indeed a reaction between HOS and individual deoxynucleosides was demonstrated. The nature of the products is currently unaer investigation. In view of the results obtained with the bioassay system, it would appear that HOS is also capable of reacting with the DNA of living cells. The effects of HOS on DNA are quite dissimilar from those that are observed when DNA is exposed to hydroxylamine, in which case neither a shift in A,,,, nor increases in buoyant densities were observed. The effects of hydroxylamine on the spectra of DNA components are also quite different from those reported llereinz5’35. The results suggest that HOS reacts with isolated DNA as well as with the DNA of living cells, as a consequence of which it may be endowed with mutagenic and oncogenic potentials. Because HOS is an industrial intermediate, it would appear that precautions against inadvertent exposure should be taken. Further studies on the chemical nature of the products of the reaction between HOS and DNA (and its components) will undoubtedly reveal whether HOS is a good model for studying carcinogenesis by aromatic amines. It must be borne in mind, however, that the aromatic portion of carcinogenic hydroxylamine-O-esters may well play a unique role in the interaction with cellular DNA. Conceivably this could invoive intercalation between DNA base pairs, a reaction which HOS could not mimic.
EFFECT OF IiO!? ON
DNA
203
ACKNOWLEDGRMENTS
The author is a Research Career Development Awardee of the Division of General Medical Sciences, U.S.P.H.S. (5K3, GM 29,024). ‘I-his investigation was supported by a gift from the Dr. George A. Carden, Jr. Special Fund for Cancer Research.
REFERENCES
4 5
6 7
8
9
10 11 12 13 14 15 16 17 18 19
20
E. C. MILLERAND J. A. MILLER,A mechanism of chemical carcinogenesis: Nature of proximate carcinogens and interactions with macromolecules, Pharmacol. Rev., 18 (1966) 805-838. J. A. MILLERAND E. C. MILLER,The metabolic activation of carcinogenic aromatic amines and amides, Progr. Exptl. Tumor Res., 11 (1969) 273-301. H. R. GIJTMANN,S. B. GALITWI AND W. A. FOLEY,The conversion of noncarcinogenic aromatic amides to carcinogenic arylhydroxamic acids by synthetic N-hydroxylation, Cancer Res., 27 (1967) 1443-1445. C. M. KING AND B. PHILLIPS, Enzyme-catalyzed reactioils of the carcinogen N-hydroxy-2fluorenylacetamide with nucleic acid, Science, 159 (1968) 1351-l 353. C. M. KING AND B. PHILLIPS, N-Hydrory-2-fluorenylacetamide: Reaction of the carcinogen with guanosine, ribonuclcic acid, deoxyribonucleic acid, and protein following enzymatic deacetylation or esteritication, J. Biol. Chem., 244 (1969) 6209-6216. J. R. DEBAUN,J. Y. R. SMITH,E. C. MILLERANDJ. A. M~~~~~,Reactivity invivoofthecarcinogen N-hydroxy-2-acetylaminofiuorene: Increase by sulfate ion, Science, 167 (1970) 184-186. J. R. DEBAUN, E. C. M~LLZRAND J. A. MILLER, N-Hydroxy-2-acetylaminofluorene sulfotransferase: Its probable role in carcinogenesis and in protein- (methir:n-S-yl) binding in rat liver, Cancer Res., 30 (1970) 577-595. V. M. MAHER,J. A. MILLER, E. C. MILLERAND W. C. SUMMERS, Mutations and loss of transforming activity of Bacillus subtilis DNA after reaction with esters of carcinogenic N-hydroxy aromatic amides. Cancer Res., 30 (1970) 1473-1480. H. S. ROSENKRANZ, Ph.D. Dissertation, Cornell University Graduate School of Medical Sciences, New York, 1959. H. K. SCHACHMAN, Ultracentrifugation, diffusion and viscometry, in S. P. Colowick and N. 0. Kaplan (Eds.), Methods ;+IEnzymology, Vol. 4, Academic Press, New York, 1957, pp. 32-103. C. L. SCH~LDKRAUT, M, MARMURAND P. Don, Determination of the base composition of deoxyribonucleic acid from its buoyant density in CsCl, J. Mol. Biol., 4 (1962) 4304t3. J. MA~MUR AND P. Dorv, Determination of the base composition of deoxyribonucleic acid from its thermal denatLration temperature, J. Mot. Biol., 5 (1962) 109-l 18. P. DELUCIA AND J. CAIRNS,lsolation of an E. coti strain with a mutation affecting DNA polymerase, Nature, 224 (1969) 1164-l 166. H. S. ROSENKRAYZ, H. S. CARRAND H. M. ROSE,Phenethyl alcohol, 1. Effect on macromolecular synthesis of Escherichfa coli, J. Bacterial., 89 (1965) 1354-1359. H. S. ROSENKRANZ,H. S. CARR AND C. MORGAN, Unusual growth properties of a bacterial strain lacking DNA polymerase, Biochem. Biophys. Res. Commun., 44 (1971) 546-549. E. E. SLATER,M. D. ANDERSON AND H. S. ROSENKRANZ,Rapid detection of mutagens and carcinogens, Cancer Res., 31 (1971) 970-973. S. J. JACOBSAND H. S. ROSENKRANZ, Detection of a reactive intermediate in the reaction between DNA and hydroxyurea, Cuncer Res., 30 (1970) 1084-1094. M. MESELSON,F. W. STAHLAND J. VINOORAD,Equilibrium sedimentation of macromolecules in density gradients, Proc. Nail. Acad. Sci. (IfS.), 43 (1957) 581-588. P. Don, J. MARMUR, J. E~NER AND C. SCHILDKRAUT,Strand separation and the specific recombination in deoxyribonucleic acids: Physical chemical studies, Proc. Natl. Acad. Sci. f&S.), 46 (1960) 461-476. K. W. KOHN AND C. L. SPEARS,Alkylated DNA: Buoyant density changes and modes of dccomposition, Biachim. Biophys. Acfa, 145 (1967) 720-733.
204 21
22
23 24
25
26 27 28 29 30
31
32 33
34 35
H. S. ROSENKRANZ G. H. BEAVER,E. R. HOLIDAYANDE. A. JOHNSON,Optical properties of nucleic acids and their components, in E. Chargaff and 1. N. Davidson (Eds.1, The Nucleic Acids, Vol. 1, Academic b, New York, 1955, pp. 493-553. R. 8. KELLY, M. R. ATKINSON,J. A. HUIWWANANDA. KORNBERG, Excisionof bone dimers and other mismatched sequences by DNA polymerase of Esc~r~chia cok, Nuture, 224 (1969) 495-501. C. D. TOWN, K. C. Sn#rr~ AND H. S. KAPLAN, DNA polymeraserequiredfor rapid repair of X-ray induced DNA strand breaks in vivu, Science, 172 (1971) 851-854. M. NAGAO AND T. SUGIMURA,Sensitivity of repair-deficient mutants and similsr mutants to 4nitroquinoline l-oxide, 4-nitropyridine l-oxide, and their derivatives, Cattcer Res., 32 (1972) 2369-2374. J. H. PHILLIPSAND D. M. BROWN,The mutagenic action of hydroxylamine, in J. N. Davidson and W. E. Cohn (Ms.). Progress fn Nucleic Acid Research and Molecular Biology. Vol. 7, Academic Press, New York, 1967, pp. 349-368. H. sc7lvsna, The reaction of tobacco mosaic virus ribonucleic acid with hydroxylamine, J. ic;3bf.Biof., 3 (1961) 447-457. H. S. R~SENKRANZ,H. S. CARR AND S. ROSNKRANZ,An alteration of the DNA isolated from ficherich~a co& treated with hydroxylamine, B&him. Biophys. A&a, 119 (1966) 434436. C. JAMONAND D. SHUGAR, Studies on possible mechanisms of hydroxylamine mutagenesis, Acta Biuchim. Polon., 15 (1968) 107-120. E. FREE%, E. B. FRFXSEAND S. GRAHAM,The oxygen-dependent reaction of hydroxylamine with nucleotides and DNA, Biochim. Biophys. Acta, 123 (1966) 17-25. H. J. RHA~SEAND E. FREES&Chemical analysis of DNA alterations, I. Base liberation and backbone breakage of DNA and oligodeoxyadenylic acid induced by hydrogen peroxide and hydroxylamine, Biochim. Biophys. Acta, 155 (1968) 476-490. H. J. RH+.=~, E. FREEZEAND M. S. MELZER, Chemicalanalysis of DNA alterations, Il. Alteration and liberation of bases of deoxynucleotides and deoxynucleosides induced by hydrogen peroxide and hydroxykmine, Biachim. Bbphvs. Acta, 155 (1968) 491-504. H. OMRA AND K. YAMAGUCHI, Action of hydroxyiamine on deoxyribonucleic acid in vitro, Enzymobgia, 33 (1%7) l-!8. P. D. LAWLEY,Reaction of hy~oxyl~ine at high #n~ntrat~on with deoxycytidine or with polycytidylic acid: Evidence that substitution of amino group in cytosine residues by hydroxylamino is a p:imary reaction, and the possible relevance to hydroxytamine mutagenesis, J. Mol. Biol.. 24 (1967) 75-81. A. T. TAYMR, S. B. GUST AND 0. W. JONES,Alteration of native DNA transcription by the mutagen hydroxylamine, Cancer Res., 30 (1970) 95-99. H. FRAENKEL-CONRAT AND B. SIN(~ER.The chemical basis for the mutagenicity of hydroxylamine and methoxyamine, Biochim. Biophys. Acta, 262 (1972) 264-268.
HYDROXYLAMINE-0-SULFONK IN V1VO REACTION WITH DNA
in The Netherlands
195
ACID: IN VITRO AND POSSIBLE
HERBERT S. ROSENKRANZ Department of Microbiology, College of Pitysicians and Surgeons, Columbia University, New York, N. Y. 10032 (U.S. A.) (Received March 9th, 1973) (Revision received June Filth, 1973) (Accepted June l&h, 1973)
SUMMARY
Exposure of DNA solutions to low levels (2 - 10’V3M)of hydroxylaminc-Osulfonic acid (HOS) resulted in limited degradation accompanied by incwases in the buoyant density of the DNA. The thermal helix-coil profile of ahe DNA was not changed significantly fo!lowing exposure to HOS. Upon thermal denaturation, the treated DNA specimens exhibited buoyant density values similar to those of heated control DNA. This is taken to mean that an added function or a modified DNA base is removed upon heating. Exposure of DNA to elevated levels of HOS (,z= 0.2 M) resulted in extensive degradation which was accompanied by spectral changes: a hypercluomic shift and an increase in the wavelength of maximum absorbance. Exposure of individual deoxynucleosides to HOS also resulted in spectral changes and in the detection of new reaction products by paper chromatographic means. HOS preferentially inhibited the growth of a bacterial strain deficient in DNA polymerase. This is a property also exhibited by known mutagens and carcinogens. This is taken to mean that HOS is capable of rescting with the DNA of living cdis as well.
INTRODUCI’ION
The carcinogenicity of aromatic arnines and amides depends upon their metabolic transform? tion to the corresponding N-hydroxy derivatives’ -3. More recently it was shown that a further N-hydroxy esterification (e.g. to the corresponding sulfate) yieided the ultimate carcinogen 4-0. The present report deals with some biol Abbreviation:HOZ, hydroxylamine-O-sulfonic acid.
196
H. S. ROsENKRANZ
in vitro effects of hydroxylamine-0-sulfonic
acid (H,N-0-SO,H; MOS). This study was undertaken to determine whether this compound could serve as a model for understanding the chemical basis of carcinogenesis by aromatic amides. METHODS
Physicochemical analysis
Sedimentation coefficients were determined in a Spinco Model E analytical ultracentrifuge equipped with an uitraviolet optical system. Solutions of DNA (0.003 % in 0.15 M NaCl containing G.015 M sodium citrate) were spun at 50 740 rev./min and pictures were taken at 2-min intervals. The photographs were traced with a Joyce-Loebl Mark III B microdensitometer and sedimentation coefficients were calculated by a modification’ of the procedure of SCHACHMAN”. The banding properties of DNA in gradients of cesium chloride were determined as described by SCHILDKRAUTet al.“. Portions of the DNA together with a reference sample (Micrococcus lysodeikticus DNA, 1.731 g/cm’ for undenatured DNA and Clostridium perfringens DNA, 1.694 g/cm3 for thermally denatured DNA) were placed in a cesium chloride solution (density, 1.70 g/ml) and centrifuged at 44 770 rev./min for 24 h. The bands formed by the specimens at their equilibrium positions were photographed and traced as above. Thermal transition profiles were measured with a Beckman DU spectrophotometer according to the manner described by MARMUR AND DoTY’~. The DNA solutions were made up in 0.015 M NaCl containing 0.0015
M sodium citrate.
The midpoint (7’,,,) of the thermal helix-coil transition curve was used to characterize the specimens. Thermal denaturation was carried out by heating samples in 0.15 M NaCl containing 0.015 M sodium citrate at l??’ for 10 min and immersing them in an ice bath. Spectra of DNA and deoxynuclecside solutions were taken with a Cary Model 11 recording spectrophotometer, the absorbance at selected wavelengths was checked with a Beckman DU-2 spectrophotometer. Corresponding pairs of specimens were diluted to the same extent for direct comparison.
Microbial assay for potential carcinogens and mutagens Portions (0.1 ml) of overnight cultures of Escherichia coli W3110 (pal A+) and its DNA polymerase-deficient derivative E. coli ~3478 (pol A;) (ref. 13) in
medium HA supplemented with 5 klg thymine per m114g15,were spread onto the surface of petri plates containing 25 ml of 1.5 “/, agar dissolved in the same medium. When the surface of the agar had dried, paper discs (6.35 mm in diameter) impregnated with the substance to be tested were deposited at the center of the plates. The plates were incubated at 37” in the dark for 14 h whereupon the diameters of the zones of inhibition were determined. Each substance was tested in replicate on several occasions. A detailed description of the procedure cau be found elsewhcrei6.
EFFECT OF HOS
ON
DNA
197
RESULTS
Reaction between DNA and HOS Exposure of DNA solutions to HOS led to a limited degradation of the polydeoxynucleotide (Table I). This depolymerization was dependent upon the temperature of incubation (Table I). It is interesting that upon thermal separation of the DNA strands the sedimentation coefficients of HOS-treated DNA did not drop precipitously (Table I); this indicates that there were not many additional “hidden” singlestrand breaks. (On the other hand, DNA exposed to hydroxyurea exhibited greatly diminished sedimentation coefficients upon thermal denaturationl’, thereby indicating the presencp of many “hidden” single-strand breaks stabilized by the doublestranded structure.) Even though the sedimentation coefficients of treated DNA decreased, this wzs not accompanied by drastic changes in the thermal helix-coil transition behavior of the treated DNA (Table I). It was, however, paralleled by significant and reproducible increases in the buoyant densities of treated DNA (Table I and Fig. I). Since it has already been shown that HOS-treated DNA was not denatured (i.e. the T, was essentially unchanged), it must be concluded that the increase in buoyant density refiected chemical modification of the DNA exposed to HOS. An examination of the tracings of the DNA bands at equilibrium (Fig. i), reveals a gradual broadening of the width of the bands. This can be taken to reflect decreased molecular weights” and it supports the sedimentation velocity data (Table I). Upon thermal denaturation the control DNA exhibited the increase in buoyant density (Table 1) associated with strand separationlg. It is however very interesting that following thermal ienaturation HOS-treated DNA’s exhibited buoyant densities nearly identical to that of the heated control DNA (Table I). Since prior to heating there was a maximal difference in buoyant densities of 0.005 g/cm” between treated and untreated DNA, it would have been expected that this difference would have been maintained or even been magnified following thermal denaturation. Because this expectation was not fulfilled it may be hypothesized either that the functional group that had been attached to the DNA was heat-labile or that heating TABLE I EFFECT OF HYDROXYLAMINE-0-SULFON~C ACID ON THE PHYSICAL CHEMICAL PROPERTlESPF DNA
Calf thymus DNA t-1 mg per ml of 0.1 M phosphate buffer, pH 7.4) was incubated with 0.0019 M HOS at the temperatures indicated for 19 h, whereupon the DNA specimens were chilled and precipitated with ethanol. The DNA fibers were washed extensively with ethanol and redissolved in 0.015 M NaCl containing 0.015 M sodium citrate. -~ . ..- -- .---.----________ .I.__-..__..___.. _---- - __.__._~_. -_-_Sedimentation Coefficien Is ( S) --..-~~_----__ Before After denaturation denaturation p__.____p ____ ___
Temperature of incubation (“C) ---.--.-
Control 4 23 37
17.1 16.3 12.2 9.3
15.0 12.6 8.3 8.0
Buoyant Densit,‘es (g/cm3) ._. _.-._-. ~-After Before denaturation denaturation ~----- -
1.704 1.704 1.706 1.709
I .722 1.723 1.721 1.722
PC)
69.6 69.8 68.8 68.1
H. S. RCX3ENKRANZ
Fig. 1. Buoyant densities of normal and HOS-treated DNA. The preparation of the specimens is described in the legend of Table I. A, Control DNA; B, C and D, .DNA exposed to 0.0019 M HOS and incubated for 19 h at 4, 23 and 37”, respectively. The band at the extreme left represent the position of the marker DNA (~~cruc~~s ~~so~e~~~cusDNA, 1.731 g/cmS). Fig. 2. Spectrum of DNA after exposure to HOS. Calf thymus DNA (I mg per ml of 0.1 Mphosphate buffer, pH 7.4) was exposed to 0.64 M HOS at 23’ for 19 h. 1, Control DNA; 2, HOS-treated DNA.
eliminated an HOS-mo~fied base from the DNA. (Thus upon thermal denaturation of alkylated DNA, 7-alkylguanine residues are split-off)**. Exposure of DNA to elevated levels of HOS (i.e. 0.64 M) resulted in degradation of the’ polydeoxynucleotide to non-sedimentable material (5.1 * IO6 g x min). owever, the spectrum of such DNA specimens was altered (Fig. 2); in addition to a hyper~hromic shift-possibly reflecting DNA denaturation2’ - there was also a shift in absorption maximum from 257.5 to 265 nm.
The effects of exposure to HOS on the absorption spectra of deoxynucleosides was inv~tigated. A comparison of the spectra of thymidine and HOS-treated thymidine (Figs. 3 and 4 and Table 11) revealed a slight decrease in absorbance of the treated material. The most drastic effect was seen at pH 13. While untreated thymidine exhibited (Fig. 3) the alkali-induced hypochromic effect and the increase in Amin previously described2 I, the HOS-treated material showed almost no effect of alkali upon the spectrum except for the ap~ara~ce of a new maximum at 216 nm (Fig. 4). Upon exposure to HOS, deoxyadenosine exhibited a hypochromic shift as well as an increase in I lllLIX at pH 7 (Fig. 5). This was paralleled by similar shifts in acid and alkali (Table la).
DNA
199
Fig. 3. Spectra of unmodified respectively.
thymidine. Curves 1, 2 and 3 represent spectra at pH 2, 7 and 13,
EFFFKT OF Has
ON
Fig 4. Spectra of HOS-modified thymidine. Thymidine (1 mg per ml of O.! M phosphate buffer, pH 7.4)was exposed to 0.24 M HOS at 54” for 90 h. The spectra of the treated material were read against blanks containing HOS. Curves I,2 and 3 represent spectra at pH 2, 7 and 13, respectively.
TABLE II SPECTRAL PROPERTIES OF DEOXYNUCLEOSIDES
HOS-treated _~_I___
Compound
il rnRZ
Lin
Deoxyadenosine
2 7 13
256 258 260
230 226 229
262 2162 261
230 230 226
Deoxyguanosine
2 7 13
253 250 266
235 223 231
248 248 245
224 225 223
Deoxycytidine
2 7 13
278 271 270
240 246 246
278 278 269
240 240 250
Thymidine
2 7 13
267 266 266
235 235 245
266 266 266 _---
235 235 236
Deoxyguanosine also showed a decreased absorbance upon exposure to HQS (compare Figs. 6 and 7). However, the most pronounced changes were decreased A,,, and &in values especially in alkali and the appearance of a new a.bsorption peak at 216 nm (Fig. 7 and Table II). Upon exposure to HOS the spectrum at neutral pH of deoxycytidine was altered drastically (Fig. 8). There was an increase in absorbance as well as an increase
Fig. 5. Spectrum of HOS-modified deoxyadenosine. The conditions of the reaction are described in the legend of Fig. 4. A, ~~~adc~~ine; B, HOS-modified d~~adenosine; pH 7.0. Fig. 6. Spectra of unmodified deoxyguanosine. Curves 1,2 and 3 repmsent spectra at pH 2,7 and 13, respectively.
Fig. 7. Spectra af HOS=modifIed daoxyguanosinc. Tttc conditions of incubation arc givon in the legend of Fig 4. Curves 1, 2 and 3 reprcsent spectra at pH 2, 7 and 13, respectively; pH 7.0. Fig, R. Spectrum of HOS-mad&d deoxycytidine. The conditians of incubation were similar to those described in the legend of trig. 4. 1, Deuxycytidine; 2, HOS-modified dcoxycytidino, pH 7,O.
in A,,,,,and Amln, Iln acid the spectra of the two preparations were quite similar while in foci they had diffe~nt Rmtn values (Figs, 9 and 10 and Table II). These spectral changes were paralleled by the appearanrx of new, as yet uni~entifi~, reaction products detectable by paper chromatography (Table III).
EFFECTOF HOS
ON DNA
Fig. 9. Spectra of unmodified deoxycytidine. Curves I, 2 and 3 represent spectra at pH 2,7 and 13, respectively. Fig. 10. Spectra of HGS-modified deoxycytidine as a function of pH. Curves 1, 2 and 3 represent spectra at oH 2, 7 and 13, respectively.
TABLE III PAPER C~R~~~~P~C
MIGRATZDt4 OF DEO~UC~~ID~
AND REACTION PRODUCTS
Solvent system: n_Butanol-Water (86: 14 v/v). Rate of m&ration
&ox_vnuciei.&ies ~~-.-Dcoxyadenosine ~oxy~~~ine Thymidine Deoxycytidine
-
Confrol
Reacted with HOS
0.31 0.15 0.49 0.20
0.41 0.37 0.56 0.39
-
_--_l__
CelIs exposed ta agents which a&et the cellular DNA attempt to repair damage
to their DNA by various repair mechanisms. One of the enzymes rvrvolved in DNA repair is DNA polymerase I (refs. 13, 22). Cells deficient in this enzyme (pal A;) have been found to be more sensitive than their pal Ai parents to the inhibitory action On the other hand, the two strains will of agents which affect cellular DNA L3*16*23. be equally sensitive to agents which affect the structure and function of other cellular components’6. Advantag: was taken of this preferential sensitivity of pot A; cells towards agents which alter cefhdar DNA to devise a simple microbian bioassay for such agents’” (see also ref. 24). In the present study it was shown that HOS inhibited the pol A; strain prcferentially (Table IV), a property it shares with the carcinogens methyl methanesulfonate and N-nitroso-IV-methylurethane, and with the mutagen mitomycin C. These agents are known to react with DNA.
H. S. ROSENKRANZ
202
On the other hand, substances known to affect cellular structures other than DNA, (e.g. methicillin, streptomycin and chloramphenicol) affected the two strains similarly (Table IV). TABLE IV EFFECT OF AGENlS ON ‘THE GROWTH OF The
DNA
POWbU3tA!&DEFlCIENT
CELLS
procedure described in the text was followed. Substances l-3 served as “positive” controls;
ia. they inhibited the pol AI- strain preferentially. Their mode of action is known to involve alteration of the cellular DNA. Substances 4-6 served as “negative” controls; they are known to affect structures other than the cellular DNA. .-
No. Agent
Amount
Diameter of zone of inhibition (mm) -..
Methyl methanelulfonate N-Nitroso-IV-methylurethane Mitomycin C Methicillin Streptomycin Chloramphenicc I HOS ______.___ .._. _-_.
-.-_.
0.13 0.02 1 30 10 30 0.14 _
ftmoles jcmoles /% Pg I% PI3 /“moles -...--__.-- --_.-__
Pol A+
Pal AI._ ___. __._
54.8 18.0 37 28.8
42.0 0 23 28.8
17
17
34 43.4
.-.
34 53.8 .._.-___.
_
DISCUSSION
Upon exposure to HOS, DNA is degraded. This depolymerization appears to result from a reaction of HOS with DNA bases as evidenced by the increased buoyant density of HOS-treated DNA. Indeed a reaction between HOS and individual deoxynucleosides was demonstrated. The nature of the products is currently unaer investigation. In view of the results obtained with the bioassay system, it would appear that HOS is also capable of reacting with the DNA of living cells. The effects of HOS on DNA are quite dissimilar from those that are observed when DNA is exposed to hydroxylamine, in which case neither a shift in A,,,, nor increases in buoyant densities were observed. The effects of hydroxylamine on the spectra of DNA components are also quite different from those reported llereinz5’35. The results suggest that HOS reacts with isolated DNA as well as with the DNA of living cells, as a consequence of which it may be endowed with mutagenic and oncogenic potentials. Because HOS is an industrial intermediate, it would appear that precautions against inadvertent exposure should be taken. Further studies on the chemical nature of the products of the reaction between HOS and DNA (and its components) will undoubtedly reveal whether HOS is a good model for studying carcinogenesis by aromatic amines. It must be borne in mind, however, that the aromatic portion of carcinogenic hydroxylamine-O-esters may well play a unique role in the interaction with cellular DNA. Conceivably this could invoive intercalation between DNA base pairs, a reaction which HOS could not mimic.
EFFECT OF IiO!? ON
DNA
203
ACKNOWLEDGRMENTS
The author is a Research Career Development Awardee of the Division of General Medical Sciences, U.S.P.H.S. (5K3, GM 29,024). ‘I-his investigation was supported by a gift from the Dr. George A. Carden, Jr. Special Fund for Cancer Research.
REFERENCES
4 5
6 7
8
9
10 11 12 13 14 15 16 17 18 19
20
E. C. MILLERAND J. A. MILLER,A mechanism of chemical carcinogenesis: Nature of proximate carcinogens and interactions with macromolecules, Pharmacol. Rev., 18 (1966) 805-838. J. A. MILLERAND E. C. MILLER,The metabolic activation of carcinogenic aromatic amines and amides, Progr. Exptl. Tumor Res., 11 (1969) 273-301. H. R. GIJTMANN,S. B. GALITWI AND W. A. FOLEY,The conversion of noncarcinogenic aromatic amides to carcinogenic arylhydroxamic acids by synthetic N-hydroxylation, Cancer Res., 27 (1967) 1443-1445. C. M. KING AND B. PHILLIPS, Enzyme-catalyzed reactioils of the carcinogen N-hydroxy-2fluorenylacetamide with nucleic acid, Science, 159 (1968) 1351-l 353. C. M. KING AND B. PHILLIPS, N-Hydrory-2-fluorenylacetamide: Reaction of the carcinogen with guanosine, ribonuclcic acid, deoxyribonucleic acid, and protein following enzymatic deacetylation or esteritication, J. Biol. Chem., 244 (1969) 6209-6216. J. R. DEBAUN,J. Y. R. SMITH,E. C. MILLERANDJ. A. M~~~~~,Reactivity invivoofthecarcinogen N-hydroxy-2-acetylaminofiuorene: Increase by sulfate ion, Science, 167 (1970) 184-186. J. R. DEBAUN, E. C. M~LLZRAND J. A. MILLER, N-Hydroxy-2-acetylaminofluorene sulfotransferase: Its probable role in carcinogenesis and in protein- (methir:n-S-yl) binding in rat liver, Cancer Res., 30 (1970) 577-595. V. M. MAHER,J. A. MILLER, E. C. MILLERAND W. C. SUMMERS, Mutations and loss of transforming activity of Bacillus subtilis DNA after reaction with esters of carcinogenic N-hydroxy aromatic amides. Cancer Res., 30 (1970) 1473-1480. H. S. ROSENKRANZ, Ph.D. Dissertation, Cornell University Graduate School of Medical Sciences, New York, 1959. H. K. SCHACHMAN, Ultracentrifugation, diffusion and viscometry, in S. P. Colowick and N. 0. Kaplan (Eds.), Methods ;+IEnzymology, Vol. 4, Academic Press, New York, 1957, pp. 32-103. C. L. SCH~LDKRAUT, M, MARMURAND P. Don, Determination of the base composition of deoxyribonucleic acid from its buoyant density in CsCl, J. Mol. Biol., 4 (1962) 4304t3. J. MA~MUR AND P. Dorv, Determination of the base composition of deoxyribonucleic acid from its thermal denatLration temperature, J. Mot. Biol., 5 (1962) 109-l 18. P. DELUCIA AND J. CAIRNS,lsolation of an E. coti strain with a mutation affecting DNA polymerase, Nature, 224 (1969) 1164-l 166. H. S. ROSENKRAYZ, H. S. CARRAND H. M. ROSE,Phenethyl alcohol, 1. Effect on macromolecular synthesis of Escherichfa coli, J. Bacterial., 89 (1965) 1354-1359. H. S. ROSENKRANZ,H. S. CARR AND C. MORGAN, Unusual growth properties of a bacterial strain lacking DNA polymerase, Biochem. Biophys. Res. Commun., 44 (1971) 546-549. E. E. SLATER,M. D. ANDERSON AND H. S. ROSENKRANZ,Rapid detection of mutagens and carcinogens, Cancer Res., 31 (1971) 970-973. S. J. JACOBSAND H. S. ROSENKRANZ, Detection of a reactive intermediate in the reaction between DNA and hydroxyurea, Cuncer Res., 30 (1970) 1084-1094. M. MESELSON,F. W. STAHLAND J. VINOORAD,Equilibrium sedimentation of macromolecules in density gradients, Proc. Nail. Acad. Sci. (IfS.), 43 (1957) 581-588. P. Don, J. MARMUR, J. E~NER AND C. SCHILDKRAUT,Strand separation and the specific recombination in deoxyribonucleic acids: Physical chemical studies, Proc. Natl. Acad. Sci. f&S.), 46 (1960) 461-476. K. W. KOHN AND C. L. SPEARS,Alkylated DNA: Buoyant density changes and modes of dccomposition, Biachim. Biophys. Acfa, 145 (1967) 720-733.
204 21
22
23 24
25
26 27 28 29 30
31
32 33
34 35
H. S. ROSENKRANZ G. H. BEAVER,E. R. HOLIDAYANDE. A. JOHNSON,Optical properties of nucleic acids and their components, in E. Chargaff and 1. N. Davidson (Eds.1, The Nucleic Acids, Vol. 1, Academic b, New York, 1955, pp. 493-553. R. 8. KELLY, M. R. ATKINSON,J. A. HUIWWANANDA. KORNBERG, Excisionof bone dimers and other mismatched sequences by DNA polymerase of Esc~r~chia cok, Nuture, 224 (1969) 495-501. C. D. TOWN, K. C. Sn#rr~ AND H. S. KAPLAN, DNA polymeraserequiredfor rapid repair of X-ray induced DNA strand breaks in vivu, Science, 172 (1971) 851-854. M. NAGAO AND T. SUGIMURA,Sensitivity of repair-deficient mutants and similsr mutants to 4nitroquinoline l-oxide, 4-nitropyridine l-oxide, and their derivatives, Cattcer Res., 32 (1972) 2369-2374. J. H. PHILLIPSAND D. M. BROWN,The mutagenic action of hydroxylamine, in J. N. Davidson and W. E. Cohn (Ms.). Progress fn Nucleic Acid Research and Molecular Biology. Vol. 7, Academic Press, New York, 1967, pp. 349-368. H. sc7lvsna, The reaction of tobacco mosaic virus ribonucleic acid with hydroxylamine, J. ic;3bf.Biof., 3 (1961) 447-457. H. S. R~SENKRANZ,H. S. CARR AND S. ROSNKRANZ,An alteration of the DNA isolated from ficherich~a co& treated with hydroxylamine, B&him. Biophys. A&a, 119 (1966) 434436. C. JAMONAND D. SHUGAR, Studies on possible mechanisms of hydroxylamine mutagenesis, Acta Biuchim. Polon., 15 (1968) 107-120. E. FREE%, E. B. FRFXSEAND S. GRAHAM,The oxygen-dependent reaction of hydroxylamine with nucleotides and DNA, Biochim. Biophys. Acta, 123 (1966) 17-25. H. J. RHA~SEAND E. FREES&Chemical analysis of DNA alterations, I. Base liberation and backbone breakage of DNA and oligodeoxyadenylic acid induced by hydrogen peroxide and hydroxylamine, Biochim. Biophys. Acta, 155 (1968) 476-490. H. J. RH+.=~, E. FREEZEAND M. S. MELZER, Chemicalanalysis of DNA alterations, Il. Alteration and liberation of bases of deoxynucleotides and deoxynucleosides induced by hydrogen peroxide and hydroxykmine, Biachim. Bbphvs. Acta, 155 (1968) 491-504. H. OMRA AND K. YAMAGUCHI, Action of hydroxyiamine on deoxyribonucleic acid in vitro, Enzymobgia, 33 (1%7) l-!8. P. D. LAWLEY,Reaction of hy~oxyl~ine at high #n~ntrat~on with deoxycytidine or with polycytidylic acid: Evidence that substitution of amino group in cytosine residues by hydroxylamino is a p:imary reaction, and the possible relevance to hydroxytamine mutagenesis, J. Mol. Biol.. 24 (1967) 75-81. A. T. TAYMR, S. B. GUST AND 0. W. JONES,Alteration of native DNA transcription by the mutagen hydroxylamine, Cancer Res., 30 (1970) 95-99. H. FRAENKEL-CONRAT AND B. SIN(~ER.The chemical basis for the mutagenicity of hydroxylamine and methoxyamine, Biochim. Biophys. Acta, 262 (1972) 264-268.