Inactivating DNA alterations induced by peroxides and peroxide-producing agents

Mutation Research Elsevier Publishing Company, Amsterdam Printed in The Netherlands

517

INACTIVATING DNA ALTERATIONS INDUCED BY PEROXIDES AND PEROXIDE-PRODUCING AGENTS

E L I S A B E T H BAUTZ F R E E S E , JIMMY GERSON*, H A R R Y TABER**, HANS-JI~RGEN R H A E S E AND ERNST F R E E S E

Laboratory of Molecular Biology, National Institute of Neurological Diseases and Blindness, National Institutes of Health,[ Public Health Service, U. S. Department of Health, Educatio~ and Welfare, Bethesda, Md. (U.S.A.) (Received February I3th, i967)

SUMMARY

Agents containing a frce-NOH group such as hydroxylamine, N-methylhydroxylamine, hydroxyurea, hydroxyurethan, and hydrazines produce H202 on exposure to oxygen. All these agents, H202 itself, and disuccinyl peroxide, predominantly induce inactivating DNA alterations, whereas their mutagenic effect (per lethal hit) on transforming DNA is small. Chemicals containing NH~ in place of NOH, such as urea and urethan (= ethylcarbamate) do not inactivate DNA. Formaldehyde and its oxidation products, formic acid, performic acid and di-hydroxymethyl peroxide do not inactivate or mutate transforming DNA at a significant rate.

INTRODUCTION

Chemical alterations of DNA have been operationally divided into mutagenic and inactivating alterations 1.. Both alterations can give rise eventually to cellular mutations but on the whole, inactivating alterations are more frequently lethal for the cell than mutagenic ones. Furthermore, the mechanism of mutation induction is quite different for the two alterations. Whereas mutagenic DNA alterations do not prevent DNA replication across the altered site and give rise to point mutations, inactivating alterations do block DNA replication and produce mostly chromosomal breaks or large chromosomal aberrations, except when they are repaired or when replication occasionally proceeds across the alteration giving rise to a point mutation. This paper shows by means of transforming DNA that agents .which produce H20,, H202 itself, and certain organic peroxides mainly inflict inactivating DNA alterations. This effect apparently involves the intermediate formation of radicals because it is inhibited by chelating agents or radical scavengers. * Present address: Jefferson Medical College of Philadelphia, Penn. (U.S.A.). ** Present address: Laboratoire de Photosynth~se, CNRS, Gif-sur-Yvette, (Seine-et-Oise) France.

Mutation Res., 4 (1967) 517-531

518

E. BAUTZ FREESE

et al.

MATERIALS AND METHODS

Transformation. DNA donor strain: 60009 ( = S B 19 Romig) = prototroph. Recipient strain: 60087 = tryptophan-. Transformation to tryptophan independence and production of fluorescent mutants were assayed as described previously 14. DNA was isolated as described by FREESE AND FREESE1~. Treatment of DNA. DNA (IOO #g/ml) in 2 M NaC1 was diluted Io-fold in the ice cold reaction mixture, mixed well, and a control sample diluted 5o-fold into ice cold stopping mixture (0.05 M Tris + 5 #g/ml catalase). All reaction mixtures were buffered by 0.02 M sodium phosphate, pH 7.5, except when other pH's are stated. The reaction tube was placed in a water bath at the desired temperature and after different times, atiquots were diluted 5o-fold into the ice cold stopping mixture. For transformation the DNA was diluted another io-fold. All points of a given curve were determined using the same batch of both DNA and transformable bacteria. Production of hydrogen peroxide was measured by the titanium sulfate method (for detail see FREESE AND FREESElS). Chemicals. Most of the chemicals were prepared by the Aldrich Chemical Company, Milwaukee, Wisconsin: N-methylhydroxylamine, O-methylhydroxylanfine = O-methoxylamine, carboxymethoxyamine, N-hydroxyurethan = ethyl N-hydroxycarbonate, hydroxyurea and N-methyl-N-nitro-N-nitrosoguanidine and nitrosopiperidine. Urethan ~ ethylurethan = ethyl-carbamate, hydrazine, hydrogen peroxide and formic acid were bought from Fisher Scientific Co. Formaldehyde was also purchased from this Company as a 3o% solution in 15% methanol; it was obtained in pure form by fractional distillation at 45 °, with a --7 °° trap after the condensing column, and collected in deionized and distilled water. Hydroxylamine HC1 was bought from the Fluka Chemical Co., Buchs, Switzerland, urea from J. T. Baker, Chem. Co., peroxidase from Sigma Chem. Co., and catalase from Worthington Biochem. Corp. Disuccinylperoxide was prepared by the method of CLOVERAND HOUGHTON9 and di-hydroxymethyl peroxide by the method of WlELAND AND WlNGLER4O; both compounds were 4 times recrystallized and freeze dried to remove all traces of H~O2. Performic acid was prepared by the method of HIRS 2~. RESULTS

Reactions of transforming DNA with H20~ (I) Inactivation, The inactivation of the tryptophan marker of a given preparation of transforming DNA by different H~O, concentrations (pH 7.5, 45 °) is shown in Fig. I. The inactivation rates increased with the H202 concentration. DNA prepared by different methods showed greatly different inactivation rates in H~O2. Fig. 2 shows, for example, the inactivation by H,O2 of 2 differently prepared DNA samples, of which one (prep. I) was barely inactivated by Io -2 M H20~ alone but responded strongly to the addition of FeSO4 (IO-s M). (This preparation was not used for other experiments in this paper, because even with Fe ~+ the rate of inactivation was much smaller than that observed for our ordinary DNA preparations (prep, II).) DNA prepared by our usual method did not exhibit an increased inactivaM~tation Res., 4 (1967) 517-531

INACTIVATING

DNA

ALTERATIONS INDUCED BY PEROXIDES

519

i0-I

•

~A

HT.0z

IO-Z

10-3

5XIO-ZM ~

10-4

~

I 10

0

i

I 20

t.

. I 30

MINUTES Fig. I. I n a c t i v a t i o n of t h e t r y p t o p h a n concentrations.

~F - ~

io_~l

~

°

o

~

m

0 - ~

HzOz'0-3M

~ +reso,

t-

~ 0

m a r k e r of B. sz,btilis t r a n s f o r m i n g D N A b y d i f f e r e n t H s O 8

l

HOURS

I

w

l

2

Fig. 2. I n a c t i v a t i o n of H s O = of 2 d i f f e r e n t l y p r e p a r e d D N A s a m p l e s .

Mutation Res., 4 (1967) 5 1 7 - 5 3 1

E. BAUTZFREESE et al.

520

tion rate when FeSO4 was added (Fig. 2), presumably because it contained enough trace metals. Sodium pyrophosphate (o.o5 M), KeN (IO 2 M), Fee13 (2. IO a M) and EDTA (IO-~ M) reduced the inactivation rate (see Fig. 3), whereas catalase (io mH/ml ) and peroxidase (IO m/~/ml) completely abolished the inactivation (Fig. 4). (2) Mutation. The induction of mutations by H202 was measured by the frequency of fluorescent colonies among the tryptophan independent transformants. The pP

~ "~,~ ~ ~ ~Fe C+ :3 |2XO I _N 31~..gDK ~~TNA

i0 -I > >

% u~

°%

iO-2

\

HOURS2

i

3

Fig. 3. DNA inactivation by H202: inhibition b y KCN = © ; E D T A = IB; p y r o p h o s p h a t e (PP) = A; FeC13(2"IO 3 M) = ~ ; FeC13(2.I0 - 4 M ) = A.

X

cz:m ~

\.

iO-t

i0-2

~ 0

~Peroxidose or

~

H202+k CataLos e

~

I I

-SM

t

HOURS

2

Fig. 4. D N A inactivation by H202: inhibition by catalase (io Hg/ml) and peroxidase (lO p g / m l ) .

Mulation Res., 4 (I967) 517-531

INACTIVATING

DNA

ALTERATIONS INDUCED BY PEROXIDES

521

frequency of these linked forward mutations increased barely measurably (about 0.04% mutations per lethal hit, see Table I). TABLE

I

RATEOFMUTATIONINDUCTIONPERLETHALHIT FORDIFFERENTAGENTS Agent Hydroxylamine

= HA

N-Me-HA

Molar concentration

pH

Temp. in °C

i io 2 I

7.5 7-5 7.5 7.5 7-5 7.5 6.2

7° 7° 7° 7° 7° 7° 75 75 45 45 45 45 45 60 60

IO-2

O-Me-HA Carboxymethoxyamine Hydroxyurethan

i i I io

Hydroxyurea Hydrazine Hydrogen peroxide Disuccinyl peroxide N-Methyl-N-nitroN-nitrosoguanidine

I /

2

6.2

IO z i I0-2 lO -3 i o -~ 0.05 0.05

7.5 5-7.5 7.5 7.5 7.5 5 7

Mutation % per lethal hit 6 ~o.o2

6 ~o.o2

3 3 o. i 5 ~o.°3 ~ o.ot ~ 0.02 ~
~ 0.04 ~ o. I 0.3 o. I i

H20= production by different agents Many agents react with oxygen, producing radicals, organic peroxides, and hydrogen peroxide. The hydrogen peroxide production of several agents, which

05

-

0.4

@

~

/

v 03 W O Z 0

0.2

_

A

rn < i

0

tO

60

90 MINUTES

I'10

150

180

F i g . 5. P r o d u c t i o n o f H I O ~ b y d i f f e r e n t c h e m i c a l s a t I O - 2 M c o n c e n t r a t i o n : (~, N - m e t h y l h y d r o x y l a m i n e ; II, h y d r a z i n e ; /k, h y d r o x y u r e a ; O, h y d r o x y u r e t h a n ; ID, O - m e t h y l h v d r o x y l a m i n e ; ~, carboxymethoxyamine; Y urea; e, urethan; /~, formaldehyde; IlL n i t r o s o g u a n i d i n e ; I], n i t r o s o piperidine.

Mutation Res., 4 (1967) 5 1 7 - 5 3 1

E. BAUTZ FREESE et al.

522

inactivate DNA, has been measured previously, as shall be mentioned in the discussion. For other agents hydrogen peroxide production was measured by the titanium sulfate method. Fig. 5 shows that peroxide production was observed for lO-2 M concentrations of hydrazine, hydroxyurea, N-hydroxyurethan, and N-methylhydroxylamine, but not for O-methyl-hydroxylamine, carboxymethoxyamine, urea, urethan, N-methylN-nitro-N-nitrosoguanidine, and nitrosopiperidine.

Reactions of transforming DNA with hydrogen peroxide producing agents In order to see whether there was a correlation bctwcen the production of H202

(i.e. radicals) and the reaction of transforming DNA, different agents were tested for their inactivating and mutagenic effect. R

A O-~e HA

i0-'

10-Z

10-3

I

0

I

2

t 3

HOURS

Fig. 6. Inactivation by IO-* M hydroxylamine (HA), N-methylhydroxylamine (N-Me-HA), O-methylhydroxylamine (O-Me-HA), and HA + P P (sodium pyrophosphate 0.o 5 M).

(x) Inactivation. (a) lO-2 M hydroxylamine or lO-2 M N-methyl-HA rapidly inactivated DNA whereas lO -2 M O-methyl-HA did not. The inactivation was abolished when 0. 5 M pyrophosphate was present. (b) Hydroxyurea (lO-2 and lO-1 M) inactivated DNA rapidly, while urea did not. In the presence of 0.5 M pyrophosphate hydroxyurea also did not inactivate DNA (Fig. 7)(c) lO-2 M N-hydroxyurethan inactivated DNA. whereas lO-3 M urethan or hydroxyurethan together with pyrophosphate showed no inactivation (Fig. 8). (d) lO-2 M hydrazine inactivated DNA; this effect was inhibited b y io m # / m l Mutation Res., 4 (1967) 517-531

INACTIVATING DNA ALTERATIONS INDUCED BY PEROXIDES

I i F i ~ _ _ _i _

i

523

HydroxyureoIO-;ZM+PP i

Ufeo IO~2M

-z M

l0-3 ~

0

,I 2

i

J

3

HOURS

Fig. 7. Inactivation by hydroxyurea and urea,

'ZM

> i0-~ > ~

i0-z

0

& HydroxyurethQn+ PP II

Ixyurethon IO'2M

J I

1 2

J 3

HOURS Fig. 8. Inactivation by hydroxyurethan and urethan.

catalase or :[0-3 M EDTA, but not by IO m#/ml peroxidase or 0.5 M pyrophosphate. In the presence of nitrogen some inactivation still took place but the inactivation rate later decreased (Fig. 9). Mutation Res., 4 (z967) 517-53 x

524

g. BAUTZ FREESE e~ al.

a

+Cotalase

i0 -I

\

Io-2 ' 5e + Peroxtdo

10-3

~

+PP

\HAZ only

I0_ 4

0

r

[

j _ _ _

I

2

3

HOURS Fig. 9- I n a c t i v a t i o n b y h y d r a z i n e a n d its i n h i b i t i o n b y [] c a t a l a s e ; [j E D T A ; • a n d A peroxidase.

N 2 atmosphere;

(e) lO -3 M N-methyl-N-nitro-N-nitrosoguanidine showed almost no i n a c t i v a tion of D N A and d i d not influence the i n a c t i v a t i o n b y H302. (2) Mutation. The i n d u c t i o n of fluorescent m u t a n t s has been m e a s u r e d for all agents for which i n a c t i v a t i o n was analyzed. A high r a t e of m u t a t i o n induction was o b s e r v e d w i t h I M concentrations of H A , N - m e t h y l - H A , O - m e t h y l - H A a n d c a r b o x y m e t h o x y a m i n e . A t concentrations of lO -2 M, however, at which m a n y agents b o t h p r o d u c e d p e r o x i d e a n d i n a c t i v a t e d D N A at a high rate, the m u t a t i o n i n d u c t i o n per lethal hit was v e r y low (see T a b l e I).

Reactions of transforming DNA with formaldehyde and organic peroxides (a) F o r m a l d e h y d e at c o n c e n t r a t i o n s of IO-1 a n d lO -2 M did not i n a c t i v a t e D N A . A m i x t u r e of lO -2 M f o r m a l d e h y d e a n d lO -1 M H302 i n c u b a t e d at 45 ° for i h, t h e n catalase a d d e d for ½ h to d e s t r o y the u n r e a c t e d H303, also did n o t i n a c t i v a t e D N A . Finally, lO -3 M H202 inactivated D N A at t h e s a m e r a t e w h e t h e r lO -3 M foi~maldehyde h a d been a d d e d or not (see Fig. IO). (b) F o r m i c acid and performic acid (lO -1 or lO -5 M) did not i n a c t i v a t e DNA. Mutation Res., 4 (I967) 5 t 7 - 5 3 1

INACTIVATING

DNA

ALTERATIONS INDUCED BY PEROXIDES

525

(c) Di-hydroxymethyl peroxide (lO -1 or I M) did not inactivate DNA (Fig. IO), if it had been sufficiently purified (4 × recrystallization + freeze drying). (After 2 × recrystallization some residual inactivation, which soon leveled off, had still been oh-

Formoldh.lO- IM ÷ ¢atala~

>

i0-l H202 F + H202

10-2

I

I

0

t

I

t

•

t

2

I

3

HOURS Fig. IO. Absence of D N A inactivation b y formaldehyde • and its oxidation p r o d u c t s : forlnic acid (FA) fl; performic acid (PFA) [l; and d i - h y d r o x y m e t h y l peroxide (DMP) O.

~DSP 10"4M " ~ . . . . _ . . . . . lO-I

4------DSP IO-3M

+ FeSO4 to-2

i0-3

I L

I

2 HOURS

I

3

Fig. I I . I n a c t i v a t i o n of D N A b y disuccinyl peroxide (DSP): O, D S P lO .3 M ; O, D S P IO-s M + FeSO, lO-5 M ; i , D S P IO-4 M ; A, D S P io-* M + FeSO 4 io -~ M.

Mutation Res., 4 (1967) 517-531

526

E.

i .-c}--cr--c--~

m

BAUTZ

FREESE et al.

'+EDTA

-I'KCN

i -,

! 10-3M i0-2

I0-3

+pp

10-4I 0

,

l l

,

l 2

,

I 3

HOURS

Fig. I2. Inhibition of DNA inactivation by disuccinyl peroxide (DSP) : inhibition by KCN and EDTA.

served, apparently caused by H202 which had been left over from the reaction of formaldehyde with H,02.) (d) Disuccinyl peroxide (lO-3 M or lO-4 M) inactivated DNA at a rate which was not influenced by the addition of FeSO4 (Fig. II). The inactivation could not be inhibited by pyrophosphate, FeC13, peroxidase, or catalase (catalase and albumin had the same small effect). However, EDTA and KCN prevented the reaction (Figs. 12 and 13). Disuccinyl peroxide at lO-3 M did not induce forward mutations at a significant rate (see Table I). DISCUSSION

Reactions with transforming D N A We have shown that several agents containing free NOH groups and hydrazine produce, in the presence of oxygen, hydrogen peroxide. Earlier papers have analyzed the production of hydrogen peroxide by isoniazid (4-NC3H,. CO" N H . NH,, a hydrazine derivative) 5°, methylhydrazine compounds4,% and by hydroxylamines 18. An oxygen effect has been reported for the chemical reactions of hydroxylamine *° and hydrazine e with DNA or its components. All peroxide producing agents that were examined, and H,O, itself, showed a Mutation Res., 4 (1967) 517-531

INACTIVATING D N A

ALTERATIONS INDUCED BY PEROXIDES

52 7

10-I

io-2

10-3

10 -4

0

L

!

I

2

3

HOURS

Fig. 13. I n a c t i v a t i o n of D N A b y d i s u c c i n y l peroxide (DSP) in t h e presence of p e r o x i d a s e (IO/~g/ml) Z~ ; c a t a l a s e ( i o / ~ g / m l ) = [] ; or a l b u m i n (IO/~g/ml) = ©.

strong inactivating effect on transforming DNA which was accompanied by the production of only very few mutations per lethal hit (see Table I). The inactivation of transforming DNA has been earlier reported for hydrogen peroxide~,~8,32 for hydroxylamine and N-methylhydroxylaminele,18; hydrazinel°. Mutations were induced aL a high rate only by high concentrations (I M) of hydroxylamines by areaction which has previously been shown to be oxygen independent le. Hydrazine has been observed to inactivate phages more rapidly at low (lO-1 M) than at high (I M) concentrationslL presumably because hydrazine both produces and destroys radicals and H~O2, similar to hydroxylamine18. Both the peroxide production and the DNA inactivation by hydroxylamin(s were inhibited by cyanide, excess of oxidized lee3+, EDTA, pyrophosphate, or catalase. It is therefore apparent that they required the formation of radicals. Most DNA preparations could react with H~Oz without tile addition of trace metals. Some DNA preparations, however, reacted only when (reduced) Fe 2+ had been added. As a polyanion, DNA tends to be covered with metals which can be removed only by exchange with other cations. Some trace metals seem to be required therefore as catalysts for the radical reactions. Mulalion Res., 4 (1967) 517-531

528

E.

TABLE

BET\VEEN

THE

PRODUCTION

OF H202

Molar concentration

Agent

lO

-

Hydroxylamine

lO

~

N-Me-HA

io

2

0-Me-HA

--

io

2

AND

INACTIVATION

H202 production pH 9, 7 °'~

et al.

=

i

-t

--

+

+

-

--

--

HA

--

--

H yctroxyurea

+

--

10 -2

Urea

.

IO

Hydroxyurethan

"

I o -~

.

Urethan

~

Hydrazine

to

Disuccinylperoxide

peroxide

-

Nitrosoguanidine

q --

Formaldehyde e

. ~

-

~

Hydrogen

i o -a

.

--

t o -a a

BY DIFFI';RENT

Mutation pH, temp. see Table 1

Carboxymethoxyamine

2

OF ])~_

Inactivation pH 7.5, 4 5

1 o -2

5 " Io

FREESE

I I

CORRELATION AGENTS

IO

BAUTZ

-

.(>)

-

-

,

(i-)

The inactivation of DNA by hydrazine was also inhibited by catalase and EDTA but not by pyrophosphate. The inhibition of the earlier-mentioned reactions by pyrophosphate is not clear: Pyrophosphate is known to chelate certain metals, but its effect might also be due to the rupture of pyrophosphate bonds by the radicals, thus acting as a radical scavenger by decreasing the available free energy of the resulting radicals. The autoxidation of hydrazine may not be inhibited by hydrazine either because it requires trace metals that cannot be chelated by pyrophosphate or because some of the produced radicals may have too low energy to split the pyrophosphate bond but high enough energy to attack DNA. 0-methylhydroxylamine, carboxymethoxyamine, urea and urethan neither produced H~O2 nor did they inactivate transforming DNA. Formaldehyde autoxidizes slowly 48; the concentration of radicals present at any time apparently is very low because the rate of DNA inactivation is negligibly slow. This ineffectiveness of low concentrations (< io -1 M) of formaldehyde on DNA has already been reported by ZAMENHOF et al) 2. At higher concentrations (o.5 M) formaldehyde reacts with RNA (ref. II) or denatured DNA (ref. 43), forming N-hydroxymethyl groups (--NH--CH2OH) with free amino groups. Native DNA shows a very small reaction which becomes significant only at high formaldehyde concentrations (4%) and causes the inactivation of transforming DNA (ret. 52). At such high concentrations (12%) formaldehyde slowly (24 h) causes crosslinking of DNA (ref. 21). These reactions do not seem to be responsible for the in vivo effect of formaldehyde in Drosophila or Neurospora, because the effective formaldehyde concentration near the chromosome should be much too small. In fact, the in vivo mutagenic effect of formaldehyde is greatly enhanced by the addition of H~Oz to formaldehyde, indicating that some oxidation product of formaldehyde may be responsible (see SOBELS41).We have therefore examined the inactivating and mutagenic effect of oxidation products of formaldehyde ::formic acid, performic acid, and dihydroxymethyl-peroxide, finding all 3 compounds ineffective. Presumably, one of these organic peroxides, which are not attacked by catalase (that renders HzO2 ineffective), causes the in vivo reaction by enzymic conversion into an active radical. It remains open whether this radical induces mutations by direct alteration of DNA or indirectly. 2VIutation Res,,

4 (1967)

517-531

INACTIVATING DNA ALTERATIONS INDUCED BY PEROXIDES

52 9

A similar enzymic activation seems to be responsible for the effect of other organic peroxides which have not shown any effect on transforming DNA but do induce mutations or inactivate cells in vivo: di-t-butyl peroxide or cumcne peroxide (reis. 28, 24). Disuccinyl peroxide, however, seems to have a sufficiently low activation cnergy for decomposition that it is effective in vitro. The inactivation of transforming DNA by this agent has been reported already by LATARJET et al. 28 and LUZZATI et al. a2. The radical involvement is indicated by the inhibition of the inactivation by EDTA and KCN. Being an organic peroxide, catalase cannot inhibit its effect. Pyrophosphate also does not inhibit the reaction. The reason for this ineffectiveness m a y be the same as that for hydrazine mentioned above.

Effects in cellular organisms Many of the radical producing agents have been shown to be lethal to microorganisms: e.g. hydrogen peroxide inactivates Staphylococcus 51 and Escherichia coli, the latter more rapidly in a strain which lacks catalase 1. Disuccinyl peroxide inactivates E. coli 27. Mycobacterium tuberculosum is inactivated by isoniazid s° and E. coli by hydrazine ~9 and hydroxyurea 38. It has not been established whether death was caused by the alteration of DNA or some other cell components. But at least in the case of hydroxyurea a drastic alteration of bacterial DNA has been observed after prolonged exposure of the cells 39. The induction of chromosomal breaks has been shown for some of the agents: hydroxylamine and m a n y of its derivatives having free NOH groups induce chromosomal breaks in mammalian cells23,41,7. Urethan induces chromosomal alterations in Oenothera, Vicia, etc.~a, a~ and in Drosophila e", presumably by enzymic conversion to hydroxyurethan ~7 and subsequent production of radicals. Maleic hydrazide induces chromosomal breaks in Vicia faba 25. Tert.-butylhydroperoxide induces chromosomal breakage and rearrangements in Vicia faba 31. Most hydroxylamines having free NOH groups or hydrazines (see TARNOWSKI et al. ~4) and m a n y peroxides (see KOTIN AND FALK2e) are carcinogenic if they carry an organic residue, which presumably is needed to permit passage of cellular (and nuclear) membranes. Several of the radical-producing chemicals examined in this paper have been reported to be mutagenic in different organisms (in bacteria: WYss et al. 51, CHEVALLIER AND LUZZATI8, LINGENS~%8°; Neurospora: DICKEY et al. 1°, WAGNER et al. 47, JENSON et al.24; Aspergillus: VAN ARKEL45; higher plants: OEHLKERS33,a4; Drosophila: RAPOPORT35, VOGT4e, AUERBACH a, ALTENBURG ~, SOBELS4°,41; p h a g e s : FREESE e~ al.

refs. 12, 13). In Drosophila recessive lethals were measured; these could have been caused by chromosomal breaks or point mutations, whereas the induction of reverse mutations presumably was caused by point mutations in Neurospora, bacteria, and phages. In most cases the mutagenic efficiency, i.e. mutations per lethal hit has not been measured, but where it has been determined, in bacteria or phages, it was low. The treatment of Neurospora conidia 24 showed a seemingly high mutagenic efficiency, but the lethal effect m a y have been masked because the conidia were multinucleate: the different nuclei can cancel a lethal effect by crossfeeding. It is therefore not clear at this time whether most of the induced mutations were caused by occasional mutagenic DNA alterations or rather by the predominant effect of inactivating DNA alterations. Mutation Res., 4 (1967) 517-531

E. BAUTZ FREESE el al.

53 °

S i n c e m o s t of t h e a b o v e a g e n t s g i v e rise t o O H r a d i c a l s , t h e c h e m i c a l r e a c t i o n s of H202 w i t h D N A a n d its c o m p o n e n t s h a v e also b e e n e x a m i n e d a n d will b e d i s c u s s e d in d i f f e r e n t papers3~, 37. I t h a s b e e n f o u n d t h a t p y r i m i d i n e r i n g s are b r o k e n f r e q u e n t l y , all f o u r b a s e s are l i b e r a t e d less f r e q u e n t l y , a n d t h e D N A b a c k b o n e is b r o k e n as a c o n s e q u e n c e of t h e s e r e a c t i o n s . All t h e s e e f f e c t s c o n s t i t u t e i n a c t i v a t i n g D N A a l t e r a t i o n s . I n a d d i t i o n , a d e n i n e (A) is o c c a s i o n a l l y a l t e r e d t o a b a s e a n a l o g A1. T h e p o s s i b i l i t y t h a t t h i s e f f e c t c o n s t i t u t e s a m u t a g e n i c D N A a l t e r a t i o n is u n d e r i n v e s t i g a t i o n . REFERENCES I ADLER, H. J., Catalase, hydrogen peroxide and ionizing radiation, Radiation Res., Suppl. 3 (1963) 11o-129. 2 ALTENBURG, L. S., The production of mutations in Drosophila by tertiary-butyl hydroperoxide, Proc. Natl. Acad. Sci. (U.S.), 4 ° (1954) lO37-1o4o. 3 AUERBACH, C., Mutation tests on Drosophila melanogaster with aqueous solutions of formaldehyde, Am. Naturalist, 86 (1952) 330-332. 4 BERNEIS, K., M. ]~OFLER, W. BOLLAG, P. ZELLER, A. KAISER AND A. LANGEMANN, Der prooxydative Effekt tumorrhemmender Methylhydrazin-Verbindungen, Helv. Chim. Acta, 46 (1963) 2157-2167. 5 BERNEIS, K., M. KOFLER AND W. BOLLAG, The influence of chelating agents on the prooxidative effect of a hydrogen peroxide producing methylhydrazine compound, Experientia, 20 (1964) 73-74. 6 BROWN, D. M., A. D. MCNAUGHT AND P. SCHELL, The chemical basis of hydrazine mutagenesis, Biochem. Biophys. Res. Commun., 24 (1966) 967-971. 7 BORENFREUND, E., •. KRIM AND A. BENDICH, Chromosomal alterations induced by hyponitrite and hydroxylamine derivatives, J. Natl. Cancer Inst., 32 (1964) 667-679. 8 CHEVALLIER, M. l~., AND D. LUZZATI, Action mutag~ne sp¢cifique de trois peroxydes organiques sur les mutations de deux loci de E. coli 15 T- 9-13, Compt. Rend., 280 (196o) 1572-1574. 9 CLOVER, A. M., AND A. C. HOUGHTON, The action of hydrogen peroxide upon anhydrides and the formation of organic acid, peroxides and peracids, Am. Chem. J., 32 (19o4) 43-68. io DICKEY, F. H., G. H. CLELAND AND C. LOTZ, The role of organic peroxides in the induction of mutations, Proc. Natl. Acad. Sci. (U.S.), 35 (1949) 581-586. i I FRAENKEL-CONRAT, i-I., Reaction of nucleic acid with formaldehyde, Biochim. Biophys. Acta, 15 (1954) 307-309. i2 FREESE, E., E. B. FREESE AND E. BAUTZ, Hydroxylamine as a mutagenic and inactivating agent, J. Mol. Biol., 3 (1961) 133-143. 13 FREESE, E., E. I:~AUTZANn E. B. FREESE, The chemical and mutagenic specificity of hydroxylamine, Proc. Natl. Acad. Sci. (U.S.), 47 (I96I) 845 855. 14 FREESE, E., ANn H. B. STRACK, Induction of mutations in transforming DNA by hydroxylamine, Proc. Natl. Acad. Sci. (U.S.), 48 (I962) 1796-18o3. 15 FREESE, E. B., AND E. FREESE, The rate of DNA strand separation, Biochemistry, 2 (1963) 7o7 715 • i6 FREESE, E. B., AND E. FREESE, Two separable effects of hydroxylamine on transforming DNA, Proc. Natl. Acad. Sci. (U.S.), 52 (1964) 1289-1297. 17 FREESE, E. B., The effects of urethan and hydroxyurethan on transforming DNA, Genetics, 5 i (1965) 953-960. 18 FREESE, E., AND E. B. FREESE, The oxygen effect on deoxyribonucleic acid inactivation by hvdroxylaulines, Biochemistry, 4 (1965) 2419-2433. 19 F'REESE, E., AND E. ]:~. FREESE, Mutagenic and inactivating DNA alterations, Radiation Res., Suppl. 6 (1966) 97-14o. 20 FREESE, E., E. B. FREESE AND S. GRAHAM,The oxygen-dependent reaction of hydroxylamine with nucleotides and DNA, Biochim. Biophys. Acta, 123 (1966) 17-25. 21 FREIFELDER, D., AND P. V. DAVISON, Physicochemical studies on the reaction between formaldehyde and DNA, Biophys. J., 3 (1963) 49-6322 HIRS, C. H. W., The oxidation of ribonuclease with performic acid, J. Biol. Chem., 219 (1956) 611-621. 23 Hsu, T. C., AND C. E. SOMERS, Effect of 5-bromodeoxyuridine on mammalian chromosomes, Proc. Natl. Acad. Sci. (U.S.), 47 (1961) 396-4°3 24 JENSEN, K. A., J. KIRK, G. KOLMARKAND M. WESTERGAARD, Chemically induced mutations in Neurospora, Cold Spring Harbor Syrup. Quant. Biol., 16 (1951) 245-261. 2 5 KIHLMAN, B. A., Factors affecting the production of chromosome aberrations by chemicals, J. Biophys. Biochem. Cytol., 2 (1956) 543-555.

Mutation Res., 4 (1967) 517-53I

INACTIVATING

DNA ALTERATIONS INDUCED BY PEROXIDES

531

26 KOTIN, P., AND H. L. FALK, Organic peroxides, hydrogen peroxide, epoxides, and neoplasia, Radiation Res., Suppl. 3 (1963) 193-211. 27 LATARJET, R., Effects of radiation and peroxides on viral and bacterial functions linked to DNA specificity. In G. E, W. WOLSTENHOLME AND C. M. O'CONNOR (Eds.), Ciba Foundation Symposium on Io~zizing Radiations and Cell Metabolism, Little, Brown, Boston, 1956, pp. 275299. 28 LATARJET, R., N. REBEYROTTE AND P. DEMERSEMAN, Action de peroxydes organiques sur un facteur transformant du pneumocoque, in M. HAISSINSKY, (Ed.), Organic Peroxides in Radiobiology, Pergamon, Oxford, 1958 , pp. 61-72. 29 LINGENS, F., Mutagene Wirkung yon Hydrazin auf E. eoli Zellen, Naturwissenschaften, 48 (1961) 480. 3° LINGENS, E., Erzeugung biochemischer Mangelmutanten yon E. coli mit Hilfe yon Hydrazin und Hydrazinderivaten, Z. Naturforseh., Igb (1964) 151-I56. 31 LOVELESS, A., Qualitative aspects of the chemistry and biology of radiomimetic (mutagenic) substances, Nature, 167 (1951 ) 338-342 . 32 LUZZATI, D., H. SCHWEITZ, M. L. BACH AND M. 1~. CHEVALLIER, Action du peroxyde succinique sur I'ADN, J. Chim. Phys., 58 (1961) lO21-1o27. 33 OEHLKERS, F., Die Ausl6sung yon Chromosomenmutationen in der Meiosis durch Einwirkung yon Chemikalien, Z. Vererbungslehre, 81 (1943) 313-341. 34 OEHLKERS, F., Die Ausl6sung von Mutationen durch Chemikalien bei Antirrhinum majus, Z. Vererbungslehre, 87 (1956) 584-589 • 35 RAPOPORT, I. A., Carbonyl compounds and the chemical mechanism of mutations (in Russian), Compt. Rend. Acad. Sci. U.R.S.S., 54 (1946) 65-67. 36 RHAESE, H., AND E. FREESE, Chemical analysis of DNA alterations, I. Base liberation and backbone breakage of DNA and oligodeoxyadenylic acid induced by hydroxylamine, in preparation. 37 I4"HAESE, H., E. FREESE AND M. MELZER, Chemical analysis of DNA alterations, II. Alteration and liberation of bases of deoxynucleotides and deoxynucleosides, in preparation. 38 ROSENKRANZ, H. S., AND H. S. CARR, Studies with hydroxyurea, J. Bacteriol., 92 (1966) 178185. 39 ROSENKRANZ, H. S., An anomalous DNA component in hydroxyurea-treated E. coli, Biochim. Biophys. Acta, 129 (1966) 618-621. 4 ° SOBELS, F. H., Organic peroxides and mutagenic effects in Drosophila, Nature, 177 (1956) 4517-4518. 41 SOBELS, F. H., Peroxides and the induction of mutations by X-rays, ultraviolet, and formaldehyde, Radiation Res., Suppl. 3, (1963) 171-183. 42 SOMERS,C. E., AND T. C. Hsu, Chromosome damage induced by hydroxylamine in mammalian cells, Proc. Natl. Acad. Sci. (U.S.), 48 (1962) 937-943. 43 STAEHELIN, M., Reaction of tobacco mosaic virus nucleic acid with formaldehyde, Biochim. Biophys. Acta, 29 (1958) 41o-416. 44 TARNOWSKI, G. S., W. KREIS, F. A. SCHMID, J. S. CAPPUCCINOAND J. H. BURCHENAL, Effect of hydroxylamine and related compounds on growth of transplanted animal tumors, Cancer Res., 26 (1966) 1279-13Ol. 45 VAN ARKEL, G. A., Modification of Ultraviolet and Formaldehyde Mutagenesis in Aspergillus nidulans. Thesis, Utrecht, 1958. 46 VOGT, M., Mutationsausl6sung bei Drosophila durch ~thylurethan, Experientia, 4 (1948) 68-69. 47 WAGNER, R. P., C. H. HADDOX, R. FUERST AND W. R. STONE, The effect of irradiated medium, cyanide and peroxide on the mutation rate in Neurospora, Genetics, 35 (195 o) 237-248. 48 WALLING, C., Free Radicals in Solution, Wiley, New York, 1957. 49 WIELAND, H., AND A. WINGLER, Qber den Mechanismus der Oxydationsvorg~.nge V., Ann. Chem., 431 (1923) 3Ol-322. 5° WINDER, G., AND J. M. DENNENY, Metal-catalyzed auto-oxidation of isoniazid, Biochem. J., 73 (1959) 500-507. 51 WYss, O., J. B. CLARK, F. HAAS AND W. S. STONE, The role of peroxide in the biological effects of irradiated broth, J. Bacteriol., 56 (1948) 51-57. 52 ZAMENHOF,A., H. E. ALEXANDERAND G. LEIDY, Studies on the chemistry of the transforming activity, J. Exptl. Med., 98 (1953) 373-395-

Mutation Res., 4 (1967) 517-53 x