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An evaluation of tween 80 effects on the survival and DNA repair in Escherichia coli following UV or gamma irradiation
Mutation Research, 49 (1978) 1--8
© Elsevier/North-Holland Biomedical Press
AN E V A L U A T I O N OF TWEEN 80 E F F E C T S ON THE S U R V I V A L AND DNA R E P A I R IN E S C H E R I C H I A C O L I FOLLOWING UV O R GAMMA I R R A D I A T I O N *
RUEY K. CHI, J.J. SCOCCA and P.C. HUANG ** Department of Biochemistry, The Johns Hopkins University, School of Hygiene and Public Health, Baltimore, Md. 21205 (U.S.A.)
(Received 14 June 1977) (Revision received 23 August 1977) (Accepted 2 September 1977)
Summary The notion that Tween 80 may be a DNA-repair inhibitor was tested with Escherichia coli. The results indicate that cell growth, colony-forming ability,
and the rate and extent of removal of thymine-containing dimers from D N A are unchanged in the presence of Tween 80. We conclude that this detergent does not increase or diminish the effect of UV or gamma irradiation to bacteria.
The nonionic detergent, 21--26 ethosorbitan monooleate ( p o l y o x y e t h y l e n e sorbitan monosoleate, Tween 80) is used widely in industry. It is used in pharmaceutical preparations as a base for suppositories and ointments. Since it is an effective solubilizer, it is used in concentrations up to 1% in vitamin preparations. In ice cream, Tween 80 constitutes 0.46% of the dry weight. Because it retards oxidation of fats, wrapping papers for butter, for instance, contain 2.5% (w/w) of this compound. Recent studies indicated that Tween 80 might be cocarcinogenic in that it p r o m o t e d that effect of known carcinogens, although alone it had no harmful effect on cells [7,8]. The evidence was that Tween 80 inhibited the removal of
* T h i s s t u d y waS s u p p o r t e d b y N I H g r a n t s E S 2 P 0 1 0 0 4 5 4 a n d 5 T 0 1 E S 0 0 0 3 4 - 1 3 . P o r t i o n o f these r e s u l t s w a s presented b e f o r e the A m e r i c a n S o c i e t y Biological C h e m i s t r y m e e t i n g s at San Francisco 1976 (Fed. Proc., 35, 1490, Abstr. 672). ** t o w h o m t o a d d r e s s c o r r e s p o n d e n c e
ultraviolet light-induced DNA lesions b y human lymphocytes. In contrast, earlier studies by others showed that in Escherichia coli [12,17,18] and Aspergillus [15], Tween 80 provided protection against gamma irradiation, presumably b y facilitating the process of rejoining of broken DNA [18]. This investigation was undertaken to examine more closely whether Tween 80 interferes with the DNA repair mechanism and if so, at which stage is it involved. Methods and materials Escherichia coli K12-SH28, (courtesy of the late H. Berger) a mutant of K12 characterized by its prolonged uptake of thymidine was used [6]. For the study of cell growth and survival, the cultures were maintained in M9 medium at 37 ° and incubated with rotary shaking at 250 rpm. Growth in liquid media was observed by increases in turbidity as measured at 650 nm with a Coleman Junior S p e c t r o p h o t o m e t e r (path length 1 cm}, and viable counts were determined by colony-forming ability on M9-agar plates. Tween 80 (Fisher Co.) was added at various concentrations by weight and volume. Agar containing more than 5% of Tween 80 would not solidify at room temperature. Survival of cells upon irradiation was determined by colony counts after the treatment. UV irradiation was performed with a 15-W germicidal lamp (General Electric G15T8) which emitted at a dose rate of 1.2 J/m2/sec, as calibrated in this laboratory with dimethyluracil actinometry. Cells were irradiated in suspension of 5 X 107 particles/ml in M9 medium, in an open petri dish on a rotating platform, under amber room-lighting conditions. Repair incubation was done in total darkness at 37 ° . For gamma irradiation, a 137Cs gamma source emitting at 2 krad/min (courtesy of S.Y. Wang} was used. Cells were irradiated at 1 X 106 particles/ml in M9 medium in Falcon plastic tubes. After irradiation, cells were appropriately diluted for plating on Tween 80--M9 agar plates and the survival determined. Tween 80 was added either before, during or after the treatment or a combination of these stages, in the medium and agar plates. UV irradiation-induced DNA lesions were specifically measured for the possible effect of Tween 80 on repair. Cells were radiolabeled with [Me-3H]thy midine (Schwarz/Mann 45 mCi/mM) at 0.133 pg/ml in M9 medium during log phase of growth. The labeled cells were washed with M9 before irradiation and harvested b y centrifugation, with or without repair, for analyses. The cell pellets were suspended in 200 #1 of formic acid (90% ACS reagent) and hydrolyzed in sealed ignition tubes (pyrex} at 175 ° for 40 min. Following hydrolysis, the seals were opened and the contents dried under vacuum at 60 ° . The residue was resuspended in 10 pl of 0.1 N HC1 for chromatography. Thymine and thymine-UV-products were separated b y ascending chromatography on Whatman No. 1 paper at room temperature for 16--20 h using n-butanol/acetic acid/H20 (200 : 30 : 75, v/v/v) as a solvent [3,4,16]. After separation, the chromatogram was dried, 1-cm strips cut, and the tritium activity counted in a liquid-scintillation counter (counting efficiency 18%). Standards of thymine dimers were prepared b y irradiating purified thymine in the presence of acetone [10].
Results (1) Effect o f Tween 80 on growth and viability orE. coli E. coli adapted well to M9 medium containing Tween 80 at concentrations up to 10%. There was no significant difference ( F test) in the viability o f cells incubated with or w i t h o u t Tween 80 (Fig. 1). All cultures attained normal levels of growth. When growth was monitored b y turbidity measurements however, a significant effect was observed in the presence of Tween 80. For a given cell density, for instance, 5 X 109 cells/ml, the A0s0 reading for cultures without Tween 80 and with 5 or 10% Tween 80 varied from 0.80 to 0.48 or 0.30, respectively. The optical effect is more pronounced at higher cell concentration (see Fig. 2) and is reversible upon washing o u t the detergent. (2) UV-dose response Two levels of response were measured with E. coli for UV irradiation in the presence or absence of Tween 80; one measured the survival and the other DNA-lesion removal.
Id
id
-~io:
J 10 ~
IrradiQtion time (hours) Fig. 1. E f f e c t o f T w e e n 8 0 o n G r o w t h . A c u l t u r e o f E. eoli K 1 2 S H 2 9 w a s i n c u b a t e d a t 37 ° w i t h v a r y i n g c o n c e n t r a t i o n s o f T w e e n 8 0 in M9 m e d i u m . A l i q u o t s w e r e t a k e n at t h e d e s i g n a t e d t i m e s a n d p l a t e d o n M9 a g a r . C o n c e n t r a t i o n s o f T w e e n are d e n o t e d b y in this a n d s u b s e q u e n t figures as 0% (o), 0.1% (~), 1%
(C]), 5% (e) and 10% (X).
/.///
g
''~
'1
'2
'3
'4 °°6~o
'.~
'6
'7
%
F i g . 2. C o r r e l a t i o n b e t w e e n t u r b i d i t y (A 5 S 0 ) a n d v i a b l e celts f-zorn c u l t u r e s w i t h v a r y i n g c o n c e n t r a t i o n s of T w e e n 80. T u r b i d i t y was m e a s u r e d at 6 5 0 n m o n a C o l e m a n J u n i o r S p e c t r o p h o t o m e t e r a n d viable cells d e t e r m i n e d b y c o l o n y c o u n t s o n M 9 a g a r p l a t e s c o n t a i n i n g n o T w e e n 8 0 . F o r s y m b o l s , see F i g . 1 .
Tween 80 was added at three different stages of growth in respect to irradiation. Those were as follow:
before
during
after --
A
+
--
B
--
--
+
C
+
+
+
The results shown in Fig. 3 indicate that there is no detectable difference in survival among the treatments; all gave a Ds0 value of 15--20 J/m 2 {F test). The presence of Tween 80, up to 1%, during the repair incubation apparently did not affect the removal of thymine-containing dimers; t h y m i n e dimers are the major UV-induced DNA lesion. Without repair, UV treatment increased the p h o t o p r o d u c t to a level of 0.4% of the t h y m i n e compared to 0.03% w i t h o u t irradiation. After 2 h of repair incubation, the level fell to 0.16--0.18% whether Tween 80 was present or not (Table 1).
10 {3
9C 8C
&
7(:} 6C
5C
4C
8 _~ 3C
> L)
_- 2C
1 0
A
,
,
,
,
,
10
20
30
40
50
B
x~, 60
Jim 2
10
20
30
40 Jim 2
50
10 9 8
7
1
U
i
10
20
30
40 J/m 2
50
60
Fig. 3. Survival of E. coli a f t e r U V i r r a d i a t i o n . ( A ) cells w e r e p r e i n c u b a t e d f o r 2 h w i t h T w e e n 80 before irradiation, then irradiated, and plated witho u t t h e d e t e r g e n t . (B) cells w e r e i n c u b a t e d w i t h T w e e n 80 a f t e r i r r a d i a t i o n . (C) T w e e n 8 0 is p r e s e n t before, during and after the irradiation. For symbols, see Fig. 1.
6O
19 8
o
~4
\o
A 0
~ i
L
4
8
i
1~2 1~6 Krad 7 - rays
20
2~4
2~8
L~
Fig. 4. Survival of E. coli a f t e r g a m m a i r r a d i a t i o n . (A) eens t r e a t e d w i t h (D) or w i t h o u t (o) T w e e n 80 d u r i n g i r r a d i a t i o n . (B) cells w e r e a l l o w e d to r e p a i r w i t h o u t (o) or w i t h T w e e n 8 0 ( o t h e r s , see Fig. 1 for s y m b o l designation).
B 0
4
8
12
16
Kracl 7 - ruys
20
24
28
7 TABLE 1 EFFECT UV
OF TWEEN 80 ON THE REMOVAL
UV treatment Dose
Repair time (rain)
OF THYMINE-CONTAINING
Tween 80 in r e p a i r medium
DIMERS INDUCED BY
c p m in thymine-containing dimer total incorporation of thymine % (cpm)
1 2 3 4 5
0 6.5 6.5 6.5 6.5
J/m 2 J/cm 2 J/era 2 J/cm 2
0 0 120 120 120
0 0 0 0.5% 1%
0.04 0.40 0.18 0.17 0.16
(100/273340) (630/157730) (639•355440) (572/342660) (582/357730)
For m e t h o d s and m a t e r i a l s , see t e x t .
(3) Response to gamma irradiation E. coli irradiated with varying doses of gamma rays were measured for survival and for repair in the presence or absence of Tween 80. Survival was compared between cultures irradiated without and with Tween 80, b u t plated after wash on M9 agar medium in the absence of this detergent. Survival was measured b y plating irradiated cells on medium containing varying concentration of Tween 80. As shown in Fig. 4, there was no apparent effect (×2 test and analysis of variance) of Tween 80 on irradiation. The Ds0 value was calculated at a b o u t 5 krad in all cases. Discussion This study has demonstrated that Tween 80 neither enhances nor reduces irradiation damage to Escherichia coli. Strains other than K12-SH28 gave identical results in experiments not shown here. Thus, this detergent does not seem to interfere with DNA repair in prokaryotes. In view of the interference of Tween 80 with the optical properties of bacteria in suspension, care must be exercised during turbidometric measurement so that a decreased absorbance in the presence of Tween 80 is not construed as a lowered rate of growth. Tween 80 was reported to p r o m o t e growth of bacteria, fungi, slime mold and other microorganisms and recommended as a routine addition in culture medium [1,9,11,13--15], b u t there is no direct evidence that it enters the cells intact. Soil cocci [14] and Pseudomonas [9] can use Tween 80 as sole carbon source for growth. The results shown here are not in direct contradiction to the suggestion of Tween 80 being cocarcinogenic in the eukaryotes [7,8], although other recent findings have indicated that Tween 80 has no specific inhibitory effect on DNA repair in Chinese hamster ovary cells [2], human peripheral l y m p h o c y t e s and HeLa cells in culture [5]. The possibility remains that Tween 80 is synergistic in action when the entry of carcinogenic compounds into cells requires enhancement. The study of possible correlation between repair deficiency and carcinogenesis would be facilitated if effective repair inhibitors could indeed be established.
References 1 A r a i , T., S. E n o m o t o a n d S. G o t o , D e t e r m i n a t i o n o f P s e u d o m o n a s aeruginosa b y b i o c h e m i c a l t e s t m e t h o d s , J a p a n . J. M i c r o b i o l . , 1 4 ( 1 9 7 0 ) 2 8 5 - - 2 9 0 . 2 B i n e n , D., a n d A.C. O l s o n , D N A r e p l i c a t i o n in C h i n e s e h a m s t e r o v a r y cells m a d e p e r m e a b l e t o n u c l e o t i d e s b y T w e e n 8 0 t r e a t m e n t , J. Cell Biol., 6 9 ( 1 9 7 6 ) 7 3 2 - - 7 3 6 . 3 B o y c e , R . P . , a n d P. H o w a r d - F l a n d e r s , R e l e a s e o f u l t r a - v i o l e t l i g h t - i n d u c e d t h y m i n e d i m e r s f r o m D N A in Escherichia coli K 1 2 , P r o e . N a t l . A c a d . Sci. ( U . S . A . ) , 51 ( 1 9 7 6 ) 2 2 6 . 4 C a r r i e r , W . L . , a n d R . B . S e t l o w , T h e e x c i s i o n o f t h y i m i d i n e d i m e r s ( t h e d e t e c t i o n o f d i m e r s in s m a l l a m o u n t s ) , in: L. G r o s s m a n a n d K . M o l d a v e M e t h o d s in E n z y m o l o g y , ( E d s . ) , Vol. 21 A c a d e m i c Press, New York, 1971, pp. 230--237. 5 C l e a v e r , J . E . , a n d R . B . P a i n t e r , A b s e n c e o f s p e c i f i c i t y a g e n t s , c o c a r c i n o g c n s , a n d s t e r o i d s in h u m a n cells, C a n c e r Res., 3 5 ( 1 9 7 5 ) 1 7 7 3 - - 1 7 7 8 . 6 F a n g m a n , W . L . , a n d A. N o v i c k , M u t a n t b a c t e r i a s h o w i n g e f f i c i e n t u t i l i z a t i o n o f t h y m i n e , J. B a c t e r i n l . , 91 ( 1 9 6 6 ) 2 3 9 0 - - 2 3 9 1 . 7 G a u d i n , D., R . S . G r e g g a n d K . L . Y i e l d i n g , D N A r e p a i r i n h i b i t i o n : a p o s s i b l e m e c h a n i s m o f a c t i o n o f c o - c a r c i n o g e n s , B i o c h e m . B i o p h y s . Res. C o m m u n . , 4 5 ( 1 9 7 1 ) 6 3 0 - - - 6 3 6 . 8 G a u d i n , D., R.S. G r e g g a n d K . L . Y i e l d i n g , I n h i b i t i o n o f D N A r e p a i r b y c o c a r c i n o g e n s , B i o c h e m . B i o p h y s . Res. C o m m u n . , 48 ( 1 9 7 2 ) 9 4 5 - - 9 4 9 . 9 H o w e , T . G . B . , a n d J . M . W a r d , T h e u t i l i z a t i o n o f T w e e n 8 0 as c a r b o n s o u r c e b y P s e u d o m o n a s , J. G e n . Microbiol., 92 (1975) 234--235. 1 0 J e n n i n g s , B . H . , S. P a s t r a - L a n d i s a n d J . W . L e r m a n , P h o t o s e n s i t i z e d d i m e r i z a t i o n o f u r a c i l , P h o t o c h e m . Photobiol., 15 (1972) 479---491. 11 K a y , E . R . M . , T h e e f f e c t s o f T w e e n 8 0 o n t h e in v i t r o m e t a b o l i s m o f cells o f t h e E h r l i c h a s c i t e s Carc i n o m a , C a n c e r Res., 2 5 ( 1 9 6 5 ) 7 6 4 - - 7 6 8 . 1 2 M c G r a t h , R . A . , a n d R . W . Williams, R e c o n s t r u c t i o n in vivo o f i r r a d i a t e d Escherichia coli d e o x y r i b o n u c l e i c acid: t h e r e j o i n i n g o f b r o k e n p i e c e s , N a t u r e ( L o n d o n ) 2 1 2 ( 1 9 6 6 ) 4 3 4 - - 5 3 5 . 1 3 M a n g a n e i l i , R . , a n d E.S. C r o s b y , E f f e c t o f d e t e r g e n t s o n s e w a g e m i c r o o r g a n i s m s , S e w a g e I n d . Wastes, 25 ( 1 9 5 3 ) 2 6 2 - - 2 7 6 . 1 4 M i n a m i , K., I s o l a t i o n o f a soil c o c c u s c a p a b l e o f u t i l i z i n g " T w e e n 8 0 " as a sole s o u r c e o f c a r b o n , Nature (London), 181 (1958) 430--431. 1 5 S c o t t , B . R . , a n d T. A l d e r s o n . D o e s " T w e e n 8 0 " i n t e r f e r e w i t h i n d u c t i o n o f m u t a g e n i c a n d l e t h a l d a m a g e in A s p e r g i l l u s ? M i c r o b . G e n e t . Bull., 3 5 ( 1 9 7 3 ) 7. 1 6 S e t l o w , R . B . , a n d W.I. C a r r i e r , T h e d i s a p p e a r a n c e o f t h y m i n e d i m e r s f r o m D N A : a n e r r o r - c o r r e c t i n g m e c h a n i s m , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 51 ( 1 9 6 1 ) 2 2 6 - - 2 3 1 . 1 7 T i t a n i , Y., a n d Y. K a t s u r e , M e c h a n i s m o f r a d i o - p r o t e c t i v e a c t i o n o f T w e e n 8 0 o n Escherichia coli, R a d i o l s o t o p t s e , 1 7 ( 1 9 6 8 ) 1 9 - - 2 2 (in J a p a n e s e ) . 1 8 T i t a n i , Y., a n d Y. K a t s u r e , E f f e c t o f T w e e n 8 0 o n s t r a n d b r e a k s o f E s c h e r i c h i a coli D N A i r r a d i a t e d in s i t u , J. R a d i a t . Res., 11 ( 1 9 7 0 ) 9 2 - - 9 7 (in J a p a n e s e ) .
© Elsevier/North-Holland Biomedical Press
AN E V A L U A T I O N OF TWEEN 80 E F F E C T S ON THE S U R V I V A L AND DNA R E P A I R IN E S C H E R I C H I A C O L I FOLLOWING UV O R GAMMA I R R A D I A T I O N *
RUEY K. CHI, J.J. SCOCCA and P.C. HUANG ** Department of Biochemistry, The Johns Hopkins University, School of Hygiene and Public Health, Baltimore, Md. 21205 (U.S.A.)
(Received 14 June 1977) (Revision received 23 August 1977) (Accepted 2 September 1977)
Summary The notion that Tween 80 may be a DNA-repair inhibitor was tested with Escherichia coli. The results indicate that cell growth, colony-forming ability,
and the rate and extent of removal of thymine-containing dimers from D N A are unchanged in the presence of Tween 80. We conclude that this detergent does not increase or diminish the effect of UV or gamma irradiation to bacteria.
The nonionic detergent, 21--26 ethosorbitan monooleate ( p o l y o x y e t h y l e n e sorbitan monosoleate, Tween 80) is used widely in industry. It is used in pharmaceutical preparations as a base for suppositories and ointments. Since it is an effective solubilizer, it is used in concentrations up to 1% in vitamin preparations. In ice cream, Tween 80 constitutes 0.46% of the dry weight. Because it retards oxidation of fats, wrapping papers for butter, for instance, contain 2.5% (w/w) of this compound. Recent studies indicated that Tween 80 might be cocarcinogenic in that it p r o m o t e d that effect of known carcinogens, although alone it had no harmful effect on cells [7,8]. The evidence was that Tween 80 inhibited the removal of
* T h i s s t u d y waS s u p p o r t e d b y N I H g r a n t s E S 2 P 0 1 0 0 4 5 4 a n d 5 T 0 1 E S 0 0 0 3 4 - 1 3 . P o r t i o n o f these r e s u l t s w a s presented b e f o r e the A m e r i c a n S o c i e t y Biological C h e m i s t r y m e e t i n g s at San Francisco 1976 (Fed. Proc., 35, 1490, Abstr. 672). ** t o w h o m t o a d d r e s s c o r r e s p o n d e n c e
ultraviolet light-induced DNA lesions b y human lymphocytes. In contrast, earlier studies by others showed that in Escherichia coli [12,17,18] and Aspergillus [15], Tween 80 provided protection against gamma irradiation, presumably b y facilitating the process of rejoining of broken DNA [18]. This investigation was undertaken to examine more closely whether Tween 80 interferes with the DNA repair mechanism and if so, at which stage is it involved. Methods and materials Escherichia coli K12-SH28, (courtesy of the late H. Berger) a mutant of K12 characterized by its prolonged uptake of thymidine was used [6]. For the study of cell growth and survival, the cultures were maintained in M9 medium at 37 ° and incubated with rotary shaking at 250 rpm. Growth in liquid media was observed by increases in turbidity as measured at 650 nm with a Coleman Junior S p e c t r o p h o t o m e t e r (path length 1 cm}, and viable counts were determined by colony-forming ability on M9-agar plates. Tween 80 (Fisher Co.) was added at various concentrations by weight and volume. Agar containing more than 5% of Tween 80 would not solidify at room temperature. Survival of cells upon irradiation was determined by colony counts after the treatment. UV irradiation was performed with a 15-W germicidal lamp (General Electric G15T8) which emitted at a dose rate of 1.2 J/m2/sec, as calibrated in this laboratory with dimethyluracil actinometry. Cells were irradiated in suspension of 5 X 107 particles/ml in M9 medium, in an open petri dish on a rotating platform, under amber room-lighting conditions. Repair incubation was done in total darkness at 37 ° . For gamma irradiation, a 137Cs gamma source emitting at 2 krad/min (courtesy of S.Y. Wang} was used. Cells were irradiated at 1 X 106 particles/ml in M9 medium in Falcon plastic tubes. After irradiation, cells were appropriately diluted for plating on Tween 80--M9 agar plates and the survival determined. Tween 80 was added either before, during or after the treatment or a combination of these stages, in the medium and agar plates. UV irradiation-induced DNA lesions were specifically measured for the possible effect of Tween 80 on repair. Cells were radiolabeled with [Me-3H]thy midine (Schwarz/Mann 45 mCi/mM) at 0.133 pg/ml in M9 medium during log phase of growth. The labeled cells were washed with M9 before irradiation and harvested b y centrifugation, with or without repair, for analyses. The cell pellets were suspended in 200 #1 of formic acid (90% ACS reagent) and hydrolyzed in sealed ignition tubes (pyrex} at 175 ° for 40 min. Following hydrolysis, the seals were opened and the contents dried under vacuum at 60 ° . The residue was resuspended in 10 pl of 0.1 N HC1 for chromatography. Thymine and thymine-UV-products were separated b y ascending chromatography on Whatman No. 1 paper at room temperature for 16--20 h using n-butanol/acetic acid/H20 (200 : 30 : 75, v/v/v) as a solvent [3,4,16]. After separation, the chromatogram was dried, 1-cm strips cut, and the tritium activity counted in a liquid-scintillation counter (counting efficiency 18%). Standards of thymine dimers were prepared b y irradiating purified thymine in the presence of acetone [10].
Results (1) Effect o f Tween 80 on growth and viability orE. coli E. coli adapted well to M9 medium containing Tween 80 at concentrations up to 10%. There was no significant difference ( F test) in the viability o f cells incubated with or w i t h o u t Tween 80 (Fig. 1). All cultures attained normal levels of growth. When growth was monitored b y turbidity measurements however, a significant effect was observed in the presence of Tween 80. For a given cell density, for instance, 5 X 109 cells/ml, the A0s0 reading for cultures without Tween 80 and with 5 or 10% Tween 80 varied from 0.80 to 0.48 or 0.30, respectively. The optical effect is more pronounced at higher cell concentration (see Fig. 2) and is reversible upon washing o u t the detergent. (2) UV-dose response Two levels of response were measured with E. coli for UV irradiation in the presence or absence of Tween 80; one measured the survival and the other DNA-lesion removal.
Id
id
-~io:
J 10 ~
IrradiQtion time (hours) Fig. 1. E f f e c t o f T w e e n 8 0 o n G r o w t h . A c u l t u r e o f E. eoli K 1 2 S H 2 9 w a s i n c u b a t e d a t 37 ° w i t h v a r y i n g c o n c e n t r a t i o n s o f T w e e n 8 0 in M9 m e d i u m . A l i q u o t s w e r e t a k e n at t h e d e s i g n a t e d t i m e s a n d p l a t e d o n M9 a g a r . C o n c e n t r a t i o n s o f T w e e n are d e n o t e d b y in this a n d s u b s e q u e n t figures as 0% (o), 0.1% (~), 1%
(C]), 5% (e) and 10% (X).
/.///
g
''~
'1
'2
'3
'4 °°6~o
'.~
'6
'7
%
F i g . 2. C o r r e l a t i o n b e t w e e n t u r b i d i t y (A 5 S 0 ) a n d v i a b l e celts f-zorn c u l t u r e s w i t h v a r y i n g c o n c e n t r a t i o n s of T w e e n 80. T u r b i d i t y was m e a s u r e d at 6 5 0 n m o n a C o l e m a n J u n i o r S p e c t r o p h o t o m e t e r a n d viable cells d e t e r m i n e d b y c o l o n y c o u n t s o n M 9 a g a r p l a t e s c o n t a i n i n g n o T w e e n 8 0 . F o r s y m b o l s , see F i g . 1 .
Tween 80 was added at three different stages of growth in respect to irradiation. Those were as follow:
before
during
after --
A
+
--
B
--
--
+
C
+
+
+
The results shown in Fig. 3 indicate that there is no detectable difference in survival among the treatments; all gave a Ds0 value of 15--20 J/m 2 {F test). The presence of Tween 80, up to 1%, during the repair incubation apparently did not affect the removal of thymine-containing dimers; t h y m i n e dimers are the major UV-induced DNA lesion. Without repair, UV treatment increased the p h o t o p r o d u c t to a level of 0.4% of the t h y m i n e compared to 0.03% w i t h o u t irradiation. After 2 h of repair incubation, the level fell to 0.16--0.18% whether Tween 80 was present or not (Table 1).
10 {3
9C 8C
&
7(:} 6C
5C
4C
8 _~ 3C
> L)
_- 2C
1 0
A
,
,
,
,
,
10
20
30
40
50
B
x~, 60
Jim 2
10
20
30
40 Jim 2
50
10 9 8
7
1
U
i
10
20
30
40 J/m 2
50
60
Fig. 3. Survival of E. coli a f t e r U V i r r a d i a t i o n . ( A ) cells w e r e p r e i n c u b a t e d f o r 2 h w i t h T w e e n 80 before irradiation, then irradiated, and plated witho u t t h e d e t e r g e n t . (B) cells w e r e i n c u b a t e d w i t h T w e e n 80 a f t e r i r r a d i a t i o n . (C) T w e e n 8 0 is p r e s e n t before, during and after the irradiation. For symbols, see Fig. 1.
6O
19 8
o
~4
\o
A 0
~ i
L
4
8
i
1~2 1~6 Krad 7 - rays
20
2~4
2~8
L~
Fig. 4. Survival of E. coli a f t e r g a m m a i r r a d i a t i o n . (A) eens t r e a t e d w i t h (D) or w i t h o u t (o) T w e e n 80 d u r i n g i r r a d i a t i o n . (B) cells w e r e a l l o w e d to r e p a i r w i t h o u t (o) or w i t h T w e e n 8 0 ( o t h e r s , see Fig. 1 for s y m b o l designation).
B 0
4
8
12
16
Kracl 7 - ruys
20
24
28
7 TABLE 1 EFFECT UV
OF TWEEN 80 ON THE REMOVAL
UV treatment Dose
Repair time (rain)
OF THYMINE-CONTAINING
Tween 80 in r e p a i r medium
DIMERS INDUCED BY
c p m in thymine-containing dimer total incorporation of thymine % (cpm)
1 2 3 4 5
0 6.5 6.5 6.5 6.5
J/m 2 J/cm 2 J/era 2 J/cm 2
0 0 120 120 120
0 0 0 0.5% 1%
0.04 0.40 0.18 0.17 0.16
(100/273340) (630/157730) (639•355440) (572/342660) (582/357730)
For m e t h o d s and m a t e r i a l s , see t e x t .
(3) Response to gamma irradiation E. coli irradiated with varying doses of gamma rays were measured for survival and for repair in the presence or absence of Tween 80. Survival was compared between cultures irradiated without and with Tween 80, b u t plated after wash on M9 agar medium in the absence of this detergent. Survival was measured b y plating irradiated cells on medium containing varying concentration of Tween 80. As shown in Fig. 4, there was no apparent effect (×2 test and analysis of variance) of Tween 80 on irradiation. The Ds0 value was calculated at a b o u t 5 krad in all cases. Discussion This study has demonstrated that Tween 80 neither enhances nor reduces irradiation damage to Escherichia coli. Strains other than K12-SH28 gave identical results in experiments not shown here. Thus, this detergent does not seem to interfere with DNA repair in prokaryotes. In view of the interference of Tween 80 with the optical properties of bacteria in suspension, care must be exercised during turbidometric measurement so that a decreased absorbance in the presence of Tween 80 is not construed as a lowered rate of growth. Tween 80 was reported to p r o m o t e growth of bacteria, fungi, slime mold and other microorganisms and recommended as a routine addition in culture medium [1,9,11,13--15], b u t there is no direct evidence that it enters the cells intact. Soil cocci [14] and Pseudomonas [9] can use Tween 80 as sole carbon source for growth. The results shown here are not in direct contradiction to the suggestion of Tween 80 being cocarcinogenic in the eukaryotes [7,8], although other recent findings have indicated that Tween 80 has no specific inhibitory effect on DNA repair in Chinese hamster ovary cells [2], human peripheral l y m p h o c y t e s and HeLa cells in culture [5]. The possibility remains that Tween 80 is synergistic in action when the entry of carcinogenic compounds into cells requires enhancement. The study of possible correlation between repair deficiency and carcinogenesis would be facilitated if effective repair inhibitors could indeed be established.
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