Peroxide effects on survival and mutation induction in ultraviolet light exposed and photoreactivated bacteria

Mutation Research Elsevier Publishing Company, Amsterdam Printed in The Netherlands

345

P E R O X I D E E F F E C T S ON S U R V I V A L AND MUTATION I N D U C T I O N IN U L T R A V I O L E T L I G H T E X P O S E D AND P H O T O R E A C T I V A T E D B A C T E R I A

C. O. DOUDNEY Research Laboratories, Department of Genetics, Albert Einstein Medical Center, Philadelphia, Penn. (U.S.A.) (Received August ist, 1968)

SUMMARY

When UV-exposed cells of Escherichia coli strain B/r were incubated in hydrogen peroxide and yeast extract, a marked lethal effect was observed within 4 rain. Previous photoreversal treatment (eliminating most pyrimidine dimers) did not reduce the amount of killing produced by the peroxide. Similar results were observed when the cells were incubated with yeast extract previously irradiated with UV. This effect was eliminated b y the presence of the peroxide decomposing enzyme, catalase, during the UV-irradiation of the yeast extract. A slight lethal effect of peroxide-containing medium on unirradiated control cultures was seen. This was increased considerably if the cultures were exposed to photoreversing light prior to incubation with peroxide. The data suggest that some component both of the UV-light and (to a lesser extent) the "white" light from the photoreversing source induces some sublethal damage to the bacteria which renders them susceptible to the lethal effect of subsequent incubation with peroxide. The mutagenic effect of the peroxide was determined at concentrations which produce considerable killing (by utilization of induction of reversion to prototrophy in a mfd ÷ and a mfd- double auxotrophic strain of E. coli). Incubation of unirradiated cultures in peroxide-containing medium caused no measurable modification of the mutation frequency. Incubation of the UV-exposed mfd +strain with peroxide resulted in an immediate and marked decline in mutation frequency which was not seen with the mfd- strain. No increase in mutation frequency of the mfd- strain was seen with incubation in peroxide suggesting that the lethal interaction of peroxide with the UV-damaged cell does not promote in surviving bacteria an increase in mutation frequency for this type of reversion to prototrophy.

INTRODUCTION

Hydrogen peroxide has been shown to produce both effects on bacteria e. The mutagenic effect of UV-irradiated been ascribed to the production of hydrogen peroxide by the been suggested that certain UV-effects including mutagenesis

lethal and mutagenic organic substrates has radiant energy 8. It has could be dependent on

Mutation Res., 6 (1968) 345-353

346

c . o . DOUDNE'Z

a comparable induction by UV of such mutagenic agents from normal cytoplasmic components6, 7. More recently, potentiation of the lethal and mutagenic effects of UV by certain DNA-combining compounds such as acridine dyes has been reported 8& An increase in mutation frequency by these agents has been shown even after elimination of most pyrimidine dimers by photoreversing light °. It has been suggested that nmtation depends both on damage by UV to the DNA and induction of cytoplasmic nmtagenic compounds which interact with the UV-damaged DNA in the mutation process ~. In this study we compared the lethal and nmtagenic effects of organic peroxides and UV-irradiated substrates on UV-damaged bacteria. We conclude that the interaction of the peroxides with some non-photoreversible and nonlethal damage induced by UV causes considerable lethality but that neither this interaction nor other effects of the peroxide have any capacity to induce a certain class of reversions to prototrophy in auxotrophic bacteria. MATERIALS AND METHODS

A Gates "Ray-Master" UV lamp (containing a General Electric 15 W quartz mercury vapor bulb) was used. It had an output below 2800 A of 8-1o erg/nlnl 2 per sec as measured by a Hanovia UV meter located at the position of the bacterial suspension. Photoreversal treatment involved exposure of the bacteria at 8 ° to light from two 500 W projection bulbs as has been previously described 4. The techniques for growth of the bacteria, UV-exposure and determination of survival and mutation frequency with postirradiation treatment have been described previously1, 2& Briefly, the bacteria were grown with rotary agitation at 37 ° from a small inoculation to an absorbance of 0.35 measured at 660 m# by a Beckman "Spectronic 2o" spectrophotometer. Growth was arrested by putting the bacterial suspension in an ice bath. The suspension was centrifuged at 6ooo rev./min in the cold, resuspended in ice cold minimal medium, centrifuged once more and once again resuspended in ice cold minimal medium. After UV-exposure and photoreversal treatment as indicated, the bacterial suspension was diluted 3 ml with 7 ml cold minimal medium containing yeast extract and hydrogen peroxide (or containing UV-irradiated yeast extract) as indicated. The bacteria were warmed rapidly to 37 °. Incubation at 37 ° on the rotary shaker for the indicated time periods followed. The hydrogen peroxide was chemically pure and was obtained from Merck and Company. Difco yeast extract (dehydrated) was used. The yeast extract was irradiated when dissolved in minimal medium with UV light from a Gates 42o-UI UV lamp. Catalase was obtained from California Foundation for Biochemical Research, Los Angeles. The bacterial strain used for the lethality study was Escherichia coli strain B/r maintained in our stocks for a number of years. A mfd + (WU 36 IO) auxotroph and a mfd (WU 36-1o-45) auxotroph of E. coli developed by WITKINTM, both of which have the same requirements for tyrosine and leucine, were used in the mutation studies. Induction of reversion of both of these requirements was followed 1°. These auxotrophic strains were kindly furnished to us by Dr. EVELYN WITKIN. Survival was determined by plating o.I nd of the bacterial suspension after appropriate post-irradiation incubation at proper dilution on the surface of minimal agar medium supplemented with 2. 5 % nutrient broth and 20 Mg per ml of L-tyrosine and L-leucine. Mutation frequency for reversion of the tyrosine requirement was determined on minimal .~/Iulation Res., 6 (1968) 345 353

347

PEROXIDE EFFECTS ON SURVIVAL AND MUTATION INDUCTION

agar medium supplemented with 2.5 % nutrient broth and 2o #~g per ml of L-leucine. Mutation frequency for reversion of the leucine requirement was determined on minireal agar medium supplemented with 2.5% nutrient broth and 20 Pg per ml of L-tyrosine. o.I ml of an appropriate lower dilution of the bacteria was plated on these media. Incubation after plating was at 37 ~'for 3 days. Three identical agar plates were averaged for all survival and mutation data given. All operations were carried out under light from gold fluorescent bulbs only to prevent photoreactivation from the room light source. RESULTS

Lethal effects When UV-irradiated cultures of E. coli strain B/r are incubated with hydrogen peroxide in yeast extract-containing minimal medium, a marked lethal effect is observed within 2 rain (Fig. i). In the example given here, 400 erg/mm 2 of UV was given to the bacteria. This is the "critical" dose (or that dose effective in producing tile m a x i m u m possible delay in initiation of DNA replication without producing non-

c

t

tO. . . .

A

o j

o

i 10 1

lO.t

•

"l~i

UV uv

3 XlO ~2 0

I

I

i

I

i

2

4

6

8

10

POSTIRRADIATION INCUBATION (MINUTES)

3X10-2

I 0

I

1 H202

I

I

!

2 3 4 5 CONCENTRATION ( X 1 0 - 4 ° / o )

. 6

Fig. I. The effect of i n c u b a t i o n w i t h h y d r o g e n p e r o x i d e on s u r v i v a l of U V - d a m a g e d E. coli s t r a i n ]3/r. A i o ml s u s p e n s i o n w a s e x p o s e d to 4 ° sec of UV ( a b o u t 40o erg/mm2). Tw o 3 ml s a m p l e s t h e n were e i t h e r (i) held in t h e d a r k a t 8 ° for 45 rain (UV), or (2) e x p o s e d to p h o t o r e v e r s i n g l i g h t a t 8 ° for 45 rain (UV, PR). The c o n t r o l c u l t u r e ((') w a s u n i r r a d i a t e d b u t h e l d a t 8 ° i n t h e d a r k d u r i n g t h e p e r i o d t h a t t h e o t h e r c u l t u r e s were b e i n g t r e a t e d . A 3 ml s a m p l e of t h e u n i r r a d i a t e d c o n t r o l c u l t u r e w a s also e x p o s e d to p h o t o r e v e r s i n g l i g h t for 45 rain a t 8 ° (C, P R ). A ft e r i r r a d i a t i o n a n d p h o t o r e v e r s a l as i n d i c a t e d , 3 ml of each b a c t e r i a l s u s p e n s i o n c o n t a i n i n g (before i r r a d i a t i o n ) a b o u t lO9 v i a b l e o r g a n i s m s per ml was d i l u t e d b y a d d i n g 7 ml of m i n i m a l m e d i u m c o n t a i n i n g a concent r a t i o n of h y d r o g e n p e r o x i d e c a l c u l a t e d to y i e l d a 5"1°-4 9o s o l u t i o n u p o n d i l u t i o n a n d a concent r a t i o n of y e a s t e x t r a c t to y i e l d a s o l u t i o n c o n t a i n i n g 4 m g pe r ml u p o n d i l u t i o n . Fig. 2. The effect of different h y d r o g e n p e r o x i d e c o n c e n t r a t i o n s on s u r v i v a l of U V - d a m a g c d , p h o t o r e v e r s e d b a c t e r i a (UV) as c o m p a r e d to u n i r r a d i a t e d b a c t e r i a (C). The e x p e r i m e n t a l c o n d i t i o n s were as in Fig. i. e x c e p t t h a t t h e c o n c e n t r a t i o n s of h y d r o g e n p e r o x i d e w e re v a r i e d to yi e l d final c o n c e n t r a t i o n s i n d i c a t e d on t h e h o r i z o n t a l axis. The r e s u l t i n g c u l t u r e s w e re i n c u b a t e d l o rain before p l a t i n g .

Mutation Res., 6 (I968) 345-353

348

c . o . DOUDNEV

dark repairable damage preventing the recovery of replication of DNA (ref. i). This dose of UV reduces the survival of strain B/r to about 20 %. Incubation with hydrogen peroxide and yeast extract reduces survival still further (to less than I °/o of the bacteria viable before irradiation). Exposure of the UV-irradiated bacteria to photoreversing light causes an increase in survival to > 60 % of those viable before UVexposure. If these bacteria are incubated in hydrogen peroxide a marked lethal effect is observed, so that a lesser number of bacteria survive than survived with the nonphotoreversed culture. This suggests that even after photoreversal treatment the UV-exposed bacteria retain some non-photoreversible damage which renders them susceptible to the lethal effect of the peroxide. Incubation with hydrogen peroxide and yeast extract has a slight lethal effect on the non-UV-exposed bacteria. This is increased somewhat by photoreversing treatment of these bacteria, suggesting that this irradiation also produces non-lethal damage which in interaction with peroxide inactivates the bacteria. By utilizing UV-exposed and photoreversed bacteria, the effectiveness of hydrogen peroxide at various concentrations was compared with its lethal effect on

10-I

v

a=

10 -2

~z >_-

PR, YE

~

~ 10-" 10-3 ~'~C, Y E

3X10-~ I I I 1 2 3 4 5 CONCENTRATION YEAST EXTRACT ( r n g )

10 -4

l 15

I 30

" ' m l 45 bO 75 90 U V DOSE (SECONDS)

i 105

• 120

Fig. 3- The effect of different y e a s t e x t r a c t c o n c e n t r a t i o n s on s u r v i v a l of U V - d a n l a g e d , p h o t o r e v e r s e d b a c t e r i a (UV) a n d u n i r r a d i a t e d b a c t e r i a (C) b o t h i n c u b a t e d w i t h 5 . 1 o -4 % h y d r o g e n p e r o x i d e . T h e e x p e r i m e n t a l c o n d i t i o n s were as in Fig. i e x c e p t t h a t t h e c o n c e n t r a t i o n s of y e a s t e x t r a c t were v a r i e d to y i e l d final c o n c e n t r a t i o n s as i n d i c a t e d on t h e h o r i z o n t a l axis. The b a c t e r i a were i n c u b a t e d i o m i n before p l a t i n g . Fig. 4- The effect of different UV-doses on s u r v i v a l of U V - d a i n a g e d a n d p h o t o r e v e r s e d b a c t e r i a i n c u b a t e d w i t h h y d r o g e n p e r o x i d e . Different c u l t u r e s a m p l e s w e re g i v e n t h e UV-doses i n d i c a t e d on t h e h o r i z o n t a l axis. E a c h i r r a d i a t e d s a m p l e was t h e n d i v i d e d i n t o 2 p a r t s a n d e i t h e r (~) e x p o s e d to p h o t o r e v e r s i n g l i g h t a t 8 ° for 45 nlin (PR) or held in t h e d a r k a t 8 ° for 45 rain (C). A 3 ml s a m p l e of each of t h e s e s u s p e n s i o n s was t h e n e i t h e r (I) d i l u t e d i n t o 7 nil of m i n i m a l m e d i u m c o n t a i n i n g sufficient h y d r o g e n p e r o x i d e to y i e l d a final c o n c e n t r a t i o n of 5 ' IO 4 % a n d sufficient y e a s t e x t r a c t to yield a final c o n c e n t r a t i o n of 4 nlg per ml a n d i n c u b a t e d for i o rain before p l a t i n g (YE) or (2) so d i l u t e d in p e r o x i d e - f r e e m e d i u m a n d p l a t e d i m m e d i a t e l y (open a n d closed circles).

Mutation Res., 6 (1968) 345-353

349

PEROXIDE EFFECTS ON SURVIVAL AND MUTATION INDUCTION

unirradiated bacteria. Concentrations of H~O2 above 2.5" lO _4 % increase killing very little in both cases (Fig. 2). That the lethal effect is dependent on yeast extract is demonstrated by the lack of response to peroxide of UV-exposed and photoreversed bacteria incubated in minimal medium not containing yeast extract (Fig. 3). Presumably, organic peroxides are formed from some component or components of the yeast extract. With this concentration of peroxide, about 3 mg/ml of yeast is required for maximum effectiveness. The effect of incubation of bacteria given various UV-doses for IO min before plating in medium containing 4 mg/ml of yeast extract and 5.1o -4 o~ /o hydrogen peroxide was compared to the unincubated controls (Fig. 4). While photoreversal treatment produced the typical and expected reduction in killing, these cultures incubated in the peroxide and yeast extract-containing medium showed at all doses a greatly reduced level of survival comparable to that shown by the UV-exposed but non-photoreversed culture. Thus it seems reasonable that these UV-exposed bacteria have suffered non-photoreversible and non-lethal damage at all UV-dose levels tested which however renders them susceptible to the lethal effect of the peroxide. The ability of UV-irradiated organic substrates to produce such lethal effects on UV-damaged bacteria was tested utilizing bacteria given 45 sec of UV and then photoreversal treatment (Fig. 5). Irradiation of the yeast extract for about 12 min produced a comparable response to that seen with hydrogen peroxide. The effect was prevented by catalase (Table I) confirming that peroxide production by the radiant energy is responsible for the effect observed. Yeast extract exposed in this manner to 1.O

A

1

°

.

-

"-,,

'° 'r

10.1

O

'%~, >

Q ~'%O

N 10_2

10_~

'~,,C O

, ,b%

•

"%

PR,YE

J 'b,C,YE

'b 10-3

i 5 10 15 IRRADIATION OF YEAST EXTRACT (MINUTES)

10 -3

I 15

I 30

I I I * 45 60 75 90 UV DOSE (SECONDS)

'

•

105

120

Fig. 5- The l e t h a l effect of i r r a d i a t e d y e a s t e x t r a c t on U V - d a m a g e d a n d p h o t o r e v e r s e d b a c t e r i a . The b a c t e r i a were t r e a t e d as d e s c r i b e d in Fig. i e x c e p t t h a t i o ml s a m p l e s of t h e y e a s t e x t r a c t s o l u t i o n (io m g / m l in m i n i m a l m e d i u m ) h a d been e x p o s e d a t a d i s t a n c e of 20 c m to t h e G a t e s 4 2 o - U i U V - l a m p for t h e periods i n d i c a t e d on t h e h o r i z o n t a l a x i s (i ns t e a d of h a v i n g h y d r o g e n p e r o x i d e added). 3 ml of t h e b a c t e r i a l s u s p e n s i o n w a s a d d e d t o 7 ml of t h e y e a s t e x t r a c t s o l u t i o n s so t r e a t e d before i n c u b a t i o n . I n c u b a t i o n w a s for i o m i n (bl a c k circles) or 20 rain (white circles) before p l a t i n g . Fig. 6. T h e effect of different UV-doses on s u r v i v a l of U V - d a m a g e d a n d p h o t o r e v e r s e d b a c t e r i a i n c u b a t e d w i t h i r r a d i a t e d y e a s t e x t r a c t . T h e e x p e r i m e n t w a s as t h a t d e s c r i b e d in Fig. 4 e x c e p t t h a t t h e y e a s t e x t r a c t s o l u t i o n w a s i r r a d i a t e d 12 m i n as d e s c r i b e d in Fig. 5 i n s t e a d of h a v i n g h y d r o g e n p e r o x i d e a d d e d to it.

Mutation Res., 6 (1968) 345-3=3

35 °

c.o.

DOUDNEY

UV produces a comparable effect on survival at all UV doses studied to that seen with hydrogen peroxide and yeast extract (Fig. 6). Thus the possibility must be suspected that UV-exposure of bacteria also results in the induction intracellularly of such organic peroxides which then interact with nonphotoreversible UV-damage to produce some lethal and possibly mutagenic effects. TABLE

I

TIlE

EFFECT

AND

PHOTOREVERSED

OF

CATALASE

ON

LETHAL

DAMAGE

OF

IRRADIATED

YEAST

EXTRACT

TO

[~V-ExPOSED

BACTERIA

Treatment

Dilution

Unirradiated, no incubation UV-exposed, no incubation UV-exposed, photorcversed, UV-cxposed, photoreversed, irradiated yeast extract UV-exposed, photoreversed, irradiated yeast extract plus

1o " io 5 1o "

IO2

I .O

131 58

[.3.10 1 5.7.io z

io 4

270

2.6" l o 2

io "

56

5 . 5 " 1o 1

no incubation i n c u b a t e d i o r a i n in i n c u b a t e d i o r a i n in catalase a

Colonies per plate

Surviving fractio*z

a C a t a l a s c ( b o v i n e l i v e r , A g r a d e , s p e c . a c t i v . 2 9 0 0 0 e n z y n i e u n i t s p e r nag p r o t e i n ) w a s a d d c d t o t h e y e a s t e x t r a c t s o l u t i o n b e f o r e i r r a d i a t i o n t o a c o n c e n t r a t i o n of i o o / , g p e r m l .

TABLE THE

II

EFFECT

IN AN

OF PEROXIDE

UNIRRADIATED

AND

H¢fd

Treallne~H ~

Unincubated

IRRADIATED

STRAIN

OF /~.

YEAST

EXTRACT

ON YIELD

OF PROTOTROPHIC

Mulalion b

Survival

lOilution

Colonies per plate

io ~

146

Surviving t?'aclion

lCeversion

Dilution

leu +

I IO

/l'r +

Incubated irradiated

lo rain, yeast extract

ro 6

98

0.67

leu ~ (yr ~

to 6

92

0.03

leu+ lI'r ~

-6"olo~ies perplate I1

~

8

I

5

lo 4

3

t ~o 1 t

~o 8 4

IO

I n c u b a t e d i o r a i n , },east extract plus H202

MUTATIONS

coli

1

2

i io t I to 1

io 8 6 4

a T h e u n i r r a d i a t e d b a c t e r i a w e r e d i l u t e d 3 inI of b a c t e r i a i n t o 7 lnl of m i n i m a l m e d i u m c o n t a i n i n g t h e p r o p e r s u p p l e m e n t of h y d r o l y s e d caseinS, =, t o w h i c h h a d b e e n a d d e d a m o u n t s of y e a s t e x t r a c t , h y d r o g e n p e r o x i d e o r i r r a d i a t e d y e a s t e x t r a c t t o g i v e t h e f i n a l c o n c e n t r a t i o n s m e n t i o n e d in F i g s . 4 a n d 6. B o t h t h e p r e i r r a d i a t i o n a n d p o s t i r r a d i a t i o n l i q u i d g r o w t h m e d i u m w a s s u p p l e m e n t e d w i t h 2 o / , g p e r mI of L - l e u c i n e a n d L - t y r o s i n e . b M u t a t i o n f r e q u e n c y w a s a l s o d e t e r m i n e d f o r t h e I0 2 d i l u t i o n a n d w a s i d e n t i c a l w i t h t h e i 0 1 d i l u t i o n . T h u s i t c a n b e c o n c l u d e d t h a t m o s t if n o t all of t h e s e c o l o n i e s a r i s e f r o m m u t a t i o n s which occur during growth on the plates. The fact that hydrogen peroxide (and irradiated yeast e x t r a c t ) d o n o t i n c r e a s e t h i s f r e q u e n c y i n d i c a t e s a l a c k of a n y l a r g e m u t a g e n i c e f f e c t b u t d o e s n o t r u l e o u t i n d u c t i o n of m u t a t i o n s a t a v e r y l o w f r e q u e n c y . T h e s e d a t a a r e r e q u i r e d f o r t h e d e m o n s t r a t i o n t h a t m o d i f i c a t i o n ill m u t a t i o n f r e q u e n c y w i t h U V - d a m a g e d b a c t e r i a ( T a b l e s l l I a n d IV) are radiation related. Incubation either with irradiated yeast extract or yeast extract plus H202 f o r 2 0 r a i n p r o d u c e d n o c h a n g e in f r e q u e n c y .

3/Iutation Res., 6 (1968) 3 4 5 - 3 5 3

PEROXIDE

351

E F F E C T S ON S U R V I V A L A N D M U T A T I O N I N D U C T I O N

Mutagenic effects The effect of hydrogen peroxide combined with yeast extract and of UVirradiated yeast extract on mutation frequency response of 2 diauxotrophic strains of E. coli identical except for a single genetic difference determining capacity for the mutation frequency decline process 1° was determined. The genetic changes followed were mutation from autotrophy to prototrophy for two genetic requirements for the amino acids, leucine and tyrosine. This allowed a comparison of the response of 2 separate mutational events TM. With the rnfd- strain (lacking capacity for the mutation frequency decline process with both of these requirements) no effect either of irradiated yeast extract-containing medium or of hydrogen peroxide added to yeast extract-containing medium was seen. This was true either with unirradiated bacteria (Table II) or with bacteria exposed to UV (Table III). Incubation of the UV-exposed mfd + strain led to a marked decline in mutation frequency within IO rain incubation (Table IV). This effect was not seen with the rnfd- strain suggesting that the promoTABLE

III

T H E E F F E C T OF H Y D R O G E N P E R O X I D E AND I R R A D I A T E D Y E A S T E X T RA CT ON Y I E L D OF PRO T O T RO PH S FROM U V - E x P O S E D m f d - STRAIN OF E. coli R E Q U I R I N G L E U C I N E AND T Y R O S I N E

Treatment a

Survival

A

Mutatio~.~

Dilution

Colonies - p e r plate

Surviving#aetion

Reversio~z

Colonies i o - i dilution b

Unincubated

IO 5

22o

o.15

leu + lvr +

43o 311

Incubated io mln yeast extract

IO 5

251

O. i 7

Incubated 2o mm yeast extract

IO -5

325

Incubated io man yeast extract plus H202

IO ~

Incubated 20 mm yeast extract plus H202

- Induced prototrophs p e r 107 auxotroph~ 195 ° 141o

leu +

35 °

1390

~¥+

280

1120

0.22

leu + tyr +

59 ° 400

181o 123o

74

o.o5I

leu+ tvr +

91 72

123o 97 °

lO -5

80

0.055

leu + tyr +

145 lO6

18Io 132o

incubated i0 mm irradiated yeast extract

io 5

85

0.058

leu + ~'r +

iii 83

131o 980

Incubated 20 mln irradiated yeast extract

I o -a

6I

0.042

leu +

I28

21oo

0~g+

IOI

1650

a T h e b a c t e r i a w e r e e x p o s e d t o 45 sec of U V ( a b o u t 4 5 0 e r g / m m 2 ) i n i o m l l o t s in c o l d p e t r i - d i s h e s w i t h s t i r r i n g a n d t h e n d i l u t e d 3 m l of b a c t e r i a i n t o 7 m l of m i n i m a l m e d i u m c o n t a i n i n g t h e p r o p e r s u p p l e m e n t of h y d r o l y s e d c a s e i n a , z, t o w h i c h h a d b e e n a d d e d a m o u n t s of y e a s t e x t r a c t , h y d r o g e n p e r o x i d e o r i r r a d i a t e d y e a s t e x t r a c t t o g i v e t h e f i n a l c o n c e n t r a t i o n s m e n t i o n e d i n F i g s . 4 a n d 6. B o t h t h e p r e - i r r a d i a t i o n a n d p o s t - i r r a d i a t i o n l i q u i d g r o w t h m e d i u m w a s s u p p l e m e n t e d w i t h 2o p g p e r m l of L - l e u c i n e a n d L - t y r o s i n e . b T h e a c t u a l d i l u t i o n in e a c h of t h e s e c a s e s w a s a d j u s t e d t o y i e l d b e t w e e n 6 o a n d 2 5 0 c o l o n i e s p e r p l a t e in o r d e r t o a v o i d i n a c c u r a c y b e c a u s e of e i t h e r s t a t i s t i c a l e r r o r o r " c r o w d i n g e f f e c t " . T h e r e s u l t s w e r e c a l c u l a t e d t o t h e i o 1 d i l u t i o n f o r e a s e of r e p o r t i n g . T h e n u m b e r of c o l o n i e s d u e t o s p o n t a n e o u s r e v e r s i o n ( T a b l e II) o n t h e p l a t e s w a s s u b t r a c t e d b e f o r e t h i s c a l c u l a t i o n w a s m a d e . A f t e r d i l u t i o n o . i m I of e a c h s u s p e n s i o n w a s p l a t e d .

tion of the mutation frequency decline process in the mfd + strain is the mechanism by which the peroxide lowers mutation frequency. This effect would be expected to obliterate the observation of any promutagenic effect of the peroxide. However, that no such promutagenic effect is seen with the mfd- strain lacking capacity for mutation frequency decline supports the idea that, for this type of mutation, organic peroxides M u t a t i o n R e s . , 6 (1968) 3 4 5 - 3 5 3

352

c. o. DOUDNEY

formed

from yeast

extract

have little or no capacity centrations

or other products

of U V - e x p o s u r e

of o r g a n i c s u b s t r a t e s

to induce nmtation

in UV-damaged

bacteria

even at con-

that are severely lethal.

T A B L E 1V THE

EFFECT

OF H Y D R O G E N

PEROXIDE

AND

YEAST

EXTRACT

ON Y I E L D

OF P R O T O T R O P H S

FROM A UV-ExPOSED

r]gfd F S T R A I N O F ]7~*. coil R E Q U I R I N G L E U C I N E A N D T Y R O S I N E

Treatm~':l a

Survival D-iihtlion

Colo~ias par plata

5"~w~,ivi*zg /)'action

Mutation l~eversion

Colonies l o 1 dilution b

I n d u c e d proloirop~s per zo v attxotrophs

Unirradiated:

Unincubatcd

lO ~

lo 7

I n c u b a t e d 2o rain y e a s t e x t r a c t p l u s H2() 2

*o ~

54

leu* °.5~

(5) e

leu~

(5) e

(vr"

(G)e

U V-exposed :

Unincubated

Io ~

34 °

0.32

leu I

163

480

iVY +

I IO

324

I n c u b a t e d lo rain yca,;t e x t r a c t

lo 5

288

0.27

lett+ tvr +

I42 113

493 392

I n c u b a t e d 20 m i n yeast extract

1o ~

44

o..t i

leutvr +

21 () t67

49 I 38o

I n c u b a t e d io rain y e a s t e x t r a c t p l u s H2() 2 I n c u b a t e d 20 m i n y e a s t e x t r a c t p l u s H~() 2

1o s

143

o.13

leu~

17

tvr ~

8

leu~ tvr +

22

i19 56 I9O 6o

lO 5

l i0

o.i i

7

a T h e b a c t e r i a were e x p o s e d to 45 sec of UV (about 45 ° erE/ram -°) in to ml lots in cold petri dishes w i t h stirring a n d t h e n d i l u t e d 3 ml of b a c t e r i a into 7 ml of m i n i m a l m e d i u m c o n t a i n i n g t h e proper s u p p l e m e n t of h y d r o l y s e d caseinS, 2, to w h i c h h a d been a d d e d a m o u n t s of y e a s t e x t r a c t a n d h y d r o g e n peroxide to give t h e final c o n c e n t r a t i o n s m e n t i o n e d in Fig. 4- B o t h t h e p r e - i r r a d i a t i o n a n d p o s t i r r a d i a t i o n liquid g r o w t h m e d i u m w a s s u p p l e m e n t e d with 2o/*g per ml of L-leucine a n d L-tyrosine. T h e effect of irradiated y e a s t e x t r a c t was identical to t h a t seen with h y d r o g e n peroxide a d d e d to y e a s t e x t r a c t . T h e effect of h y d r o g e n peroxide a n d irradiated y e a s t e x t r a c t was also d e t e r m i n e d with U V - e x p o s e d bacteria b o t h of t h e m f d + a n d m f d s t r a i n i r r a d i a t e d with p h o t o r e v e r s i n g light. No c h a n g e in t h e o b s e r v e d f r e q u e n c y w a s o b s e r v e d either after lO or 2o rain of i n c u b a t i o n . b T h e a c t u a l dilution in each of t h e s e cases was a d j u s t e d to yield b e t w e e n 6o a n d x5 ° colonies per plate in order to avoid i n a c c u r a c y because of either statistical error or " c r o w d i n g effect". T h e results were calculated to t h e lO t dilution for ease of reporting. T h e n u m b e r of colonies d u e to s p o n t a n e o u s reversion on t h e plates was subt r a c t e d before this calculation was made. After dilution o.i ml of each s u s p e n s i o n was plated. e T h e s e v a l u e s for s p o n t a n e o u s m u t a t i o n f r e q u e n c y r e p r e s e n t m u t a t i o n s which occur w i t h g r o w t h of t h e bacteria on t h e a g a r m e d i u m after p l a t i n g a n d were d e t e r m i n e d for all t r e a t m e n t s a n d dilutions a n d s u b t r a c t e d f r o m t h e total n u m b e r of colonies with t h e irradiated b a c t e r i a before calculation of colonies per Io ~ dilution. DISCUSSION T h e p o t e n t i a t i o n of U V - e f f e c t s b y p o s t i r r a d i a t i o n t r e a t m e n t s is w e l l k n o w n . I n particular DNA-combining compounds such as certain dyes have the ability to increase mutation frequency response and lethality in UV-exposed bacteriaS.For example, the dye, acriflavine enhances

both

the lethal and nmtagenic

e f f e c t s of U V 9 a n d

p r e v e n t s h o s t cell r e a c t i v a t i o n . I t a l s o e x e r t s s o m e a c t i o n o n U V - d a m a g e d bacteria w h i c h i n t e r f e r e s w i t h R N A , D N A a n d p r o t e i n s y n t h e s i s a. W h i l e l i g h t e x p o s u r e (eliminating most pyrimidine dimers) removes a part of the dye effect on DNA synthesis, some residual non-photoreversible damage after higher UV doses remains WIulalion Res., 6 (I968) 345-353

PEROXIDE EFFECTS ON SURVIVAL AND MUTATION INDUCTION

353

which results in blockage of DNA synthesis in interaction with acriflavine ~. The lethal effect of hydrogen peroxide in yeast extract-containing medium is therefore not surprising even with UV-damaged bacteria exposed to photoreversing light which would eliminate most of the pyrimidine dimers. It seems established that the light incident from the ordinary germicidal lamp has some component wavelength(s) capable of inducing sublethal damage to bacteria which is made lethal by agents such as organic peroxides. The appropriate action spectra studies for induction of this particular type of damage have not been carried out. The failure to observe a promutagenic effect of the peroxide with UV-damaged bacteria is somewhat unexpected in view of its established mutagenicity in bacteria 6 and the increase in mutation frequency response seen with incubation with dyes such as acriflavine ~. We can conclude that organic peroxides and other products of UVirradiation of organic substrates are not able to induce in UV-damaged bacteria this sort of reversion to prototrophy. Such coparticipation in other nmtagenic processes is not precluded b y these results and can even be expected in view of the general mutagenicity of peroxides and the lethal effect on UV-damaged bacteria observed. The promotion by peroxide of the mutation frequency decline process in the mfd ÷ strain but not in the mfd strain could reflect an effect of peroxide on RNA and protein synthesis in UV-damaged bacteria. This would be expected to lower mutation frequency in the mfd+ strain only TM. ACKNOWLEDGEMENTS

Some preliminary aspects of this study were carried out in the Section of Genetics, The University of Texas M. D. Anderson Hospital and Tumor Institute, Houston. The author wishes to acknowledge the valuable technical assistance there of Miss BETTY J . BRUCE and of Miss CAROL M. THORNTON at his present institution. This investigation was supported in part b y U.S. Atomic Energy Commission contract AT (3o-1)-3893. REFERENCES 1 DOUDNEY, C. O., AND C. S. YOUNG, U l t r a v i o l e t light i n d u c e d m u t a t i o n a n d d e o x y r i b o n u c l e i c acid replication in bacteria, Genetics, 47 (I962) I I 2 5 - I I 3 8 . 2 DOUDNEY, C. O., U l t r a v i o l e t l i g h t - i n d u c e d m u t a t i o n as a n e v e n t in t h e p h y s i o l o g y of t h e bacterial cell, in F. H. SOBELS (Ed.), Repair from Genetic Radiation Damage, P e r g a m o n , L o n d o n , I963, pp. I 2 5 - I 4 9 . 3 DOUDNEY, C. O., B. F. WHITE AND B. J. BRUCE, Acriflavine modification of nucleic acid form a t i o n , m u t a t i o n i n d u c t i o n a n d s u r v i v a l in u l t r a v i o l e t light e x p o s e d bacteria, Biochem. Bit)phys. Res. Commun., 15 (r964) 70-75 . 4 DOUDNEY, C. O., P h o t o r e v e r s a l of u l t r a v i o l e t light i n d u c e d m u t a t i o n , l e t h a l i t y a n d d a m a g e to nucleic acid f o r m a t i o n in bacteria, Mutation Res., 3 (1966) 280-297. 5 DOUDNEY, C. O., P h o t o r e v e r s a l of acriflavine inhibition of d e o x y r i b o n u c l e i c acid replication in u l t r a v i o l e t l i g h t - e x p o s e d bacteria, Photochem. Photobiol., 6 (1967) 65I 656. 6 HAAS, F. L., J. CLARK, O. WYSS AND W. S. STONE, M u t a t i o n s a n d m u t a g e n i c a g e n t s ill bacteria, Am. Naturalist, L X X X I V (195 o) 261-274. 7 HAAS, F. g . , AND C. O. DOUDNEY, A relation of nucleic acid s y n t h e s i s to r a d i a t i o n induced m u t a t i o n f r e q u e n c y in bacteria, Proc. Natl. Acad. Sci. (U.S.), 43 (1957) 871-883. 8 WITKIN, E., Modification of m u t a g e n e s i s initiated b y u l t r a v i o l e t light t h r o u g h p o s t t r e a t m e n t of b a c t e r i a w i t h basic dyes, J , Cell. Comp. Physiol., (Suppl. 1), 58 (1961) 135-144. 9 WITKIN, E., T h e effect of acriflavine on p h o t o r e v e r s a l of lethal a n d m u t a g e n i e d a m a g e prod u c e d in b a c t e r i a b y U.V. light, Proc. Natl. Acad. Sci. (U.S.), 5 ° (1963) 425-43c. IO WITKIN, E., R a d i a t i o n - i n d u c e d m u t a t i o n s a n d their repair, Science, 152 (1966) 1345 1353.

Mutation Res., 6 (1968) 345-353