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DNA strand breakage caused by dichlorvos, methyl methanesulphonate and iodoacetamide in Escherichia coli and cultured chinese hamster cells
.~utation Research, 24 (1974) 365-378 © Elsevier Scientific Publishing Company, A m s t e r d a m - - P r i n t e d in The Netherlands
365
DNA STRAND B R E A K A G E CAUSED BY DICHLORVOS, M E T H Y L M E T H A N E S U L P H O N A T E AND IODOACETAMIDE IN ESCHERICHIA COLI AND C U L T U R E D C H I N E S E HAMSTER CELLS
M. H. L. GREEN, A. S. C. MEDCALF, C. F. ARLETT, S. A. HARCOURT AND A. R. LEHMANN
MRC Cell Mutation Unit, University of Sussex, Falmer, Brighton, BNr 90,G (Great Britain) (Received February 5th, 1974)
SUMMARY
The DNA damaging properties of dichlorvos (2,2 dichlorovinyl dimethyl phosphate), methyl methanesulphonate (MMS) and iodoacetamide (IAA) have been studied, using alkaline sucrose sedimentation. In a strain of E. coli deficient in DNA polymerase I (polA) both dichlorvos and MMS caused random strand breakage, MMS being about twice as efficient as dichlorvos on a molar basis. In pol + bacteria, DNA strand breaks or alkali labile bonds were detected following treatment with roughly five-fold higher concentrations of MMS but at similar high concentrations of dichlorvos there was an all or none breakdown of DNA molecules to fragments of very low molecular weight which correlated well with lethality. DNA synthesized after treatment of pol÷ and polA bacteria with MMS was of low molecular weight, indicating the presence of discontinuities. With dichlorvos, the effect was much smaller. Apparent all-or-none DNA breakdown was also found when the polA strain of E. coli was treated with low concentrations of iodoacetamide, an agent that does not detectably alkylate DNA. At higher concentrations the breakdown was suppressed and random strand breakage occurred instead. These effects did not occur with pol÷ bacteria and correlated well with the greater sensitivity to iodoacetamide of the polA strain in survival experiments. We suggest that the major DNA damage resulting from treatment with iodoacetamide and dichlorvos arises indirectly through alkylation of other cellular constituents and consequent uncontrolled nuclease attack on the DNA. Discontinuities in newly synthesized DNA and mutagenesis following dichlorvos treatment, however, presumably result from direct alkylation of DNA. Strand breakage caused by dichlorvos and MMS in Chinese hamster cells tended to correlate with the extent to which these agents alkylate DNA, but survival tended to correlate with the alkylation of protein.
Abbreviations: dichlorvos, 2,2 dichlorovinyl dimethyl phosphate; IAA, iodoacetamide; MMS, methyl methanesulphonate.
366
M.H.L.
GREEN cl ol.
INTRODUCTION
The insecticide dichh;rvos has been shown to alkylate DNA ~ and may be a potential environmental nmtagen. With regard to the types of alkylations produced in DNA dichlorvos resembles MMS more closely than other common alkylating agents although dichlorvos has the additional capacity to dimethylphosphorylate protein2L On a molar basis it is about as effective as MMS in alkylating protein but about 15-fold less potent in alkylating DNA ~2. BRI1)GES et al. ~ have compared the lethal and mutagenie effects of dichtorw~s with those of MMS in a series of radiati(m-sensitive strains of Escherichia coll. ExrA, RecA and PolA strains of E. coli show 4 increased sensitivity t o both agents. Mutagenesis with both dichhnvos and MMS occurs 1)\, misret)air (i.e. ExrA or RecA strains are not mutated). There is, however, nmch less diffeience in survival between resistant and sensitive strain, s with (tichlorvos than with MMS, an(1 dichlmvos is a nmch weaker mutagen L lake MMS'S,v,~s,'za,z6 dichlorvos causes DNA strand breakage a and we wondered if it were misrepair of such straud breaks that made these agents mutagenic. If so, the differences in nmtagenicity between dichlorvos and MMS might relate to differences in the type and number of strand breaks. In this paper we show that (tichh)rw~s and MMS do differ in the type of strand breakage they cause. We also find that dichloi vos appears to cause more damage to I)NA in E. col," than can be accounted for by direct alkylation. We have consi(lered the possibility that such damage may occur indirectly, by alkylation of other cellular constituents leading to enzymatic attack on DNA, and, in this connection we have shown that IAA, an agent that does not alkylate I)NA to a measurable extent ~°, can also cause DNA damage. Finally, we have extended our study' to include the lethal and strand-breaking effects of dichlorvos in cultured Chinese hamster cells. MATERIALS AND METHODS
Escherichia coli Strains. WP2 (UV resistant), WP67 uvrA polA, WP2 uvrA, CM56I exrA and W P I o o uvrA recA have been described previously and are approximately isogenicL We are indebted to Dr. E. M. WITKIN for supplying strains WP67 and WPIoo. Media. Growth medium was M9 salts supplemented with : Casamino acids 1% ; glucose o.5}/o • tri-sodimn citrate I mg/ml and tryptophan 25 Ftg/ml. Citrate was required for the growth nf strain WP67. Buffer was BOYLE AND SYMONDS2 phosphate buffer. Viabilities were determined on nutrient agar (Oxoid Nutrient Broth No. 2 solidified with 1.5°/i~ New Zealand Agar). Reagents. [all]methyl thymidine, 22 ('i/mmole, the Radiochemical Centre, Amersham, Bucks. Dichlorvos was kindly given to us by Mr. B. J. DEAN, Tunstall Laboratory, Shell Research, Sittingbourne, Kent. MMS, Ralph N. Emmanuel, Wembley, Middlesex. IAA, Sigma Ltd., London. Sarkosyl, Geigy Ltd., Manchester. Dulbecco's troffer A, Oxoid, London. Protocol. Overnight cultures of E. coli were diluted into glowth medium containing [aHlmethyl thymidine (5° ~Ci/ml; 0. 5/~g/ml) and IOO/~g/ml deoxyadenosine. They were incubated for about 2. 5 h, filtered and the cells resuspended in buffer. A known volume was added to a bottle containing an ethanolic solution of dichlorvos or
DICHLORVOS AND M M S INDUCED D N A STRAND BREAKS
367
an aqueous solution of IAA to give an appropriate final concentration of the agent. Stocks of dichlorvos and MMS were freshly made up and diluted in analar ethanol. The final concentration of ethanol during treatment did not exceed 20/0. IAA was freshly made up in buffer. Except where otherwise indicated, treatment was for 60 rain at 37 ° with shaking. Following treatment cells were layered directly on alkaline sucrose gradients. When repair cf initial damage was examined cells were filtered, resuspended in growth medium, incubated further at 37 ° and samples taken at intervals. To examine the molecular weight of newly-synthesized DNA, unlabelled cultures were treated, filtered and resuspended in growth medium containing 50/~Ci/ml ~3H]methyl thymidine (specific activity 22 Ci/mmole) and ioo/~g/ml deoxyadenosine. After IO rain they were filtered again and resuspended in growth medium without label. Samples were layered on alkaline sucrose gradients at intervals. It was necessary to halve the culture volume at each filtration to compensate for losses of cells on the filter. In survival experiments cells were treated with IAA in similar fashion to gradient experiments, diluted at least ioo-fold and plated on nutrient agar.
Chinese hamster The Chinese hamster cell line V79-4 was routinely maintained in Eagle's Minimal Essential Medium (MEM) with 15% foetal calf serum (Flow Laboratories) and was used during passages 7 ° to 92 . Our tissue culture techniques and materials have been described in detail elsewhere 1. Eor investigation of colony-forming ability cells in the exponential growth phase were trypsinized (0.025% for io rain) from monolayer culture and after centrifugation and resuspension in culture medium a range of dilutions was dispensed into 5 cm petri dishes (Sterilin Ltd) in 5 ml medium. After 4 h incubation to allow the cells to become attached to the surface and recommence growth, but before any increase in cellular multiplicity2L the medium was removed. Two ml of Dulbecco's buffer A was added, containing an appropriate concentration of dichlorvos or MMS. Stocks of lO% MMS and dichlorvos in ethanol were freshly prepared and diluted in buffer. The final concentration of ethanol did not exceed 0.8% in any treatment and this level of ethanol was also added to the controls. Treatment was for 30 rain at 37 °, after which the buffer was removed and replaced with 5 ml fresh medium. The treated plates were incubated for 8 days before staining with I ~o methylene blue. The criterion for survival was the production of a clone containing more than 50 cells. For the strand breakage studies, on day i cells were dispensed at I × lO 5 cells per 5 cm petri dish in 5 ml medium during the morning. At 5 p.m. the medium was supplemented with E3Hlmethyl thymidine (5 #Ci/ml; I/~M) and the plates incubated overnight. On day 2 the culture medium was removed and replaced with 2 ml Dulbecco's buffer A. Fresh ethanolic solutions of dichlorvos and MMS were added to give an appropriate final concentration. The level of ethanol did not exceed 2% in any treatment. Treatment was for 60 min at 37 °, following which the buffer was removed and replaced with 0.3 ml of Dulbecco's buffer A solution containing 0.2 g/1 E D T A ~1. The cells were scraped off with a piece of silicon rubber.
.x.L H. L. {;REEN el al.
368
Alkaline sucrose sedimentation E. coli. Samples of o.o8 ml or less were lysed in a l a y e r of o. i ml S a r k o s y l lysis m i x t u r e (Sarkosyl, I }; ; N a O H , 0. 5 M; NaC1, o.2 M ; E D T A o.oi M) on a linear g r a d i e n t of 5-2O~o (w/v) sucrose in 0.2 N N a O H , 0.5 N NaC1 a n d o.ooi M E D T A . The c o n c e n t r a t i o n of o.oi M E D T A in the lysis m i x t u r e , r a t h e r t h a n o.oo~ :11 as used p r e v i o u s l y 8 was found to give b e t t e r results. The g r a d i e n t s were spun at 27 00o r e v . / m i n for 9 ° rain in a Spinco S W 5 o . I rotor. F r a c t i o n s were collected on strips of W h a t m a n 3 MM paper, washed and c o u n t e d as described previously ". Chinese hamster. Before layering, sanlples received a dose of I.S k r a d v-irradiation in order to reduce the molecular weight sufficiently to prevent the s e d i m e n t a t i o n p r o b l e m s which occur with v e r y high m o l e c u l a r weight D N A ~3. Samples of 0.o 5 ml or less (to give less t h a n IO 4 cells per gradient) were lvsed in a lavcr of o.I ml of 2 °,/ o . s o d i u m d o d e c y l s u l p h a t e and o.02 M E D T A above a linear g r a d i e n t of a 5 - 2 o % (w/v) sucrose in o.I M NaC1 and o.I M N a O H . G r a d i e n t s were spun at 38ooo r e v . / m i n for 7 ° min a n d fractions were collected as with E. coli. Similar results were o b t a i n e d o m i t t i n g the ;,-irradiation a n d using a CsCl-sucrose shelf to collect the high molecular weight m a t e r i a h
Calculation of induced stra~zd-break freque~wies It Mo a n d M,~ are the n u m b e r average molecular-weights of the u n t r e a t e d and t r e a t e d p o p u l a t i o n s of D N A molecules, the n u m b e r of s t r a n d breaks p r o d u c e d per average u n t r e a t e d molecule is equal to
Mo/M,,-- I Molecular weights (M) of each fraction were calculated using a relationship derived from STUDIER2L M ~ D 2.5 where D is the distance s e d i m e n t e d b y molecules in t h a t fraction. Values of Mo a n d M,~ were d e t e r m i n e d from e q u a t i o n (3) in the A p p e n d i x : .14n
=
:]In'
-- M 1
where M ( is the n u m b e r - - a v e r a g e molecular weight of those molecules which had s e d i m e n t e d further t h a n the sixth fraction from the t o p of t h e gradient, a n d 3 [ t ( = 8 × IO 6) is the n o m i n a l molecular weight of molecules in the sixth fraction. Since we were only i n t e r e s t e d in relative efficiencies of s t r a n d breakage, no a t t e m p t was m a d e to c a l i b r a t e the g r a d i e n t s accurately. Mo was a p p r o x i m a t e l y its n o m i n a l value I x IO ~. RESULTS
Strand breakage in a polA strain Profiles of s t r a n d b r e a k a g e caused b y dichlorvos a n d MMS in strain W P 0 7
uvrA polA have been published p r e v i o u s l y 4. I n d u c e d s t r a n d b r e a k frequencies from a series of similar e x p e r i m e n t s were calculated, a v e r a g e d a n d p l o t t e d in Fig. I. The induction of s t r a n d b r e a k s b y MMS in strain W P 6 7 uvrA polA seemed to give a nonlinear dose-response. Nevertheless, on a m o l a r basis, dichlorvos was roughly half as efficient as MMS in inducing s t r a n d breaks in a polA strain. S t r a n d breaks could not be d e t e c t e d at low doses of MMS and dichlorw>s in a polA + strain, suggesting t h a t the b r e a k s o b s e r v e d at these doses in strain W P 6 7
DICHLORVOS
AND
MMS
INDUCED
DNA
STRAND
369
BREAKS
would, in the polA + strain, be rapidly repaired by DNA polymerase I, a situation similar to that found with X-rays 2~. If repair by DNA polymerase I is error freeL these breaks are unlikely to he a major cause of dichlorvos or MMS mutagenesis in a polA ÷ strain. Since DNA polymerase I is normally associated with the repolymerization step of strand rejoining it would seem likely that the lesions exist in the polA strain as actual breaks rather than alkali labile bonds. We cannot say whether such breaks are formed enzymatically or chemically.
Strand breakage by M M S in a polA + strain From Fig. I it can be seen that the number of MMS induced strand breaks was about 5-6-fold less in strain WP2 poIA + than in strain WP67 uvrA polA so that breaks were only detectable at higher doses in the former. Fig. 2 shows the result of an experiment in which strain WP2 was treated with o.I}/o MMS for 60 min, the MMS removed and incubation continued for a further 60 rain in a growth medium. Treatment with o.1% MMS caused a considerable initial reduction in the molecular weight of the DNA on the gradient which was partly but not completely restored during further incubation. Following treatment with 0.2% MMS little restoration of molecular weight occurred during subsequent incubation (data not shown). The reduction in molecular weight in Fig. 2 m a y represent actual breaks in the DNA in the cell or alkali labile lesions. 005 0.1 0.025 005
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Fig. I. C a l c u l a t e d i n d u c e d D N A s t r a n d b r e a k frequencies for s t r a i n s W P 6 7 uvrA polA a n d W P 2 polA + t r e a t e d w i t h d i c h l o r v o s a n d MMS. F r e q u e n c i e s are n o r m a l i s e d a g a i n s t t h e c o n t r o l n u m b e r a v e r a g e m o l e c u l a r w e i g h t (approx. i × i o 8 daltons). B a s e d on t h e a v e r a g e of 3 - 6 e x p e r i m e n t s . O, MMS polA ; 0, d i c h l o r v o s polA ; &, MMS polA +. Fig. 2. A l k a l i n e sucrose s e d i m e n t a t i o n of D N A of s t r a i n W P 2 polA + t r e a t e d w i t h MMS. O, unt r e a t e d c o n t r o l ; &, t r e a t e d w i t h o.i % MMS for 6o rain; e , t r e a t e d w i t h o.I % MMS for 60 min, r e s u s p e n d e d a n d i n c u b a t e d in g r o w t h m e d i u m for 60 min.
37 o
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D N A "catastrophe" caused by dichlorvos T r e a t m e n t of strain W P 2 with dichlorvos gave a result quite different from t h a t o b t a i n e d with MMS. I n s t e a d of a s t e a d y shift of the peak to lower molecular weight with increasing dose, the peak m o v e d d i r e c t l y from n o r m a l to w'r\, h)w molecular weight. R a t h e r t h a n r a n d o m s t r a n d breakage, it a p p e a r e d t h a t i n d i v i d u a l D N A molecules had d i s i n t e g r a t e d in an all-or-none fashion a n d a p p e a r e d in the low molecular weight position. This usually occurred when strain W P 2 was t r e a t e d with v e r y high c o n c e n t r a t i o n s of dichlorvos (between a b o u t (>.20/0 and o.4'~ , v/v)* and corresponded well with our previous o b s e r v a t i o n 4 of a sharp loss of viahilitv between these concentrations. Lower c o n c e n t r a t i o n s of dichlorw)s had no d e t e c t a b l e effect ()n DNA. An e x a m p l e of this c h r o m o s o m a l " c a t a s t r o p h e " is shown in l;ig. 3. Strain \\"P2 w a s t r e a t e d with 0.4% dichlorvos and samples t a k e n at o, Io, 2o and 4 ° rain. It can be seen t h a t the profile after IO rain represents two p o p u l a t i o n s r a t h e r t h a n a distribution of r a n d o m l y broken molecules; some D N A molecules were u n b r o k e n , others were s h a t t e r e d . A f t e r 20 rain most of the D N A h a d shifted from normal to low molecular weight. W e found no evidence t h a t dichlorvos i n d u c e d a t y p e of s t r a n d breakage similar to t h a t caused by MMS in strain W P 2 ; it only a p p e a r e d to induce c a t a s t r o p h i c c h r o m o s o m a l b r e a k d o w n . The low molecular weight m a t e r i a l was not repaired bu~ was further d e g r a d e d to acid-soluble m a t e r i a l when the cells were r e t u r n e d to growth nredium (data not shown).
Molecular weight of DNA .2vnthesized following treatment with M M S a~2d dichlorvos In order to e x a m i n e the size of newly-synthesized D N A , t r e a t e d and u n t r e a t e d cells were pulse-labelled with 3 H , t h y m i d i n e . To m a k e valid comparisons, the pulse times were a d j u s t e d so t h a t the a m o u n t of D N A synthesized in u n t r e a t e d an(l t r e a t e d cells was similar. This avoids labelling artefacts similar to those described by LV.HMANN A N D O R M E R O I ) 14 a n d ~ E D G W I C K AND B R I D G E S ~°. l:ollowing t r e a t m e n t with o.o4% MMS, D N A of low molecular weight was synthesized in strain W P 2 (Irig. 4) ~. The m o l e c u l a r weight increased to t h a t of u n t r e a t e d cells on further incubation. This situation seems to be similar to t h a t seen after U V - i r r a d i a t i o n ~'. P r e s u m a b l y the new D N A c o n t a i n e d discontinuities opposite some lesion in the p a r e n t a l strands, and these were s u b s e q u e n t l y filled in. I t was not possible to calculate a useful frequency of gaps induced in newlysynthesized D N A for several reasons. A direct calculation of n u m b e r average molecular weight would be worthless, because of the error i n t r o d u c e d bv spuriously s e d i m e n t i n g m a t e r i a l in the t o p few fractions. The correction described in the APPt.:N1~IX could not be e m p l o y e d , because the m a t e r i a l s e d i m e n t i n g further t h a n the t o p fractions did not even a p p r o x i m a t e to a r a n d o m b r e a k a g e profile. In the calculations for Fig. I a p p r o x i m a t e l y equal values for n u m b e r average molecular weight were o b t a i n e d with cut-off points (M1) of 0.08 × Mo, 0.44 / M,, or 1.32 / M,, which suggests * A l t h o u g h t h e s o l u b i l i t y of d i c h l o r v o s in w a t e r is o n l y a b o u t o. I (}i) (B. J. DEAN, pers. c o m m . ) wc c o n s i s t e n t l y o b t a i n i n c r e a s e s in e f f e c t of d i c h l o r v o s w i t h n o m i n a l c o n c e n t r a t i o n s as h i g h as o.2'>, o r o .4 o. .'o. \ ¥ h e n d i c h l o r v o s is a d d e d as a 4 o o s o l u t i o n in e t h a n o l , it is s e e n t o s e p a r a t e b u t n o t w h e n a d d e d as a 2 o % s o l u t i o n . T h e a d d i t i o n a l d i c h l o r v o s m i g h t d i s s o l v e p a r t l y in t h e e t h a n o l , o r p a r t l y in cell c o n s t i t u e n t s .
DICHLORVOS AND
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Fig. 3. Alkaline sucrose sedimentation of D N A of strain WP2 polA + treated with 0.4% dichlorvos. O, u n t r e a t e d contlol; A, treated with dichlorvos for io min; £, treated for 2o min; O, treated foz 4 ° min. Fig. 4- Alkaline sucrose sedimentation of D N A synthesized by strain W P 2 polA + after t r e a t m e n t with MMS. O, u n t r e a t e d control, IO min pulse; 4, treated with o.o5% MMS for 6o min filtered, pulse labelled for IO min, refiltered and resuspended in g r o w t h m e d i u m and sampled; e , t r e a t e d w i t h MMS, labelled, refiltered etc., incubated 60 min before sampling. The counts in the treated samples have been multiplied x 5 to allow for filtration losses.
that this method gives a good estimate of Mn. With a hyperdisperse profile, like the control in Fig. 4 the calculated number-average molecular weight would increase in an arbitrary way with the cut-off point chosen. We therefore investigated calculations which were based on the heavier fractions and which were not dependent on a control with a random breakage profile. These, however, were vitiated by another difficulty. It was evident from Fig. 4 and other experiments that some gap filling was occurring in the treated sample during the IO min pulse. This meant that the heaviest material in the treated profile did not give a true indication of the induced frequency of gaps and could not be used either. Nevertheless after a given dose of MMS the daughter strand discontinuities seemed to be greater in number than the strand breaks in parental DNA (Figs. I and 2), and seemed to be repaired more completely. With neither type of lesions did further joining occur with periods of incubation greater than 6o min. Treatment with o.o4% MMS also induced discontinuities in newly-synthesized DNA in strain WP67 uvrA polA (Fig. 5)- Though consistent the effect appears less marked because as is evident from Fig. 5 the control newly-synthesized DNA was also of low molecular weight'74 °. Again it was not possible to calculate a useful frequency for induced gaps or determine whether or not MMS caused an equal number of gaps in newly-synthesized DNA in a polA and polA + strain. Finally Fig. 6 shows that treatment with o.1% dichlorvos caused no more than a slight reduction in the molecular weight of newly-synthesized DNA in strain WP2.
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Fig. 5. Alkaline sucrose sedimentation of DNA synthesized by strain \VP07 u v r A p o l A after t r e a t m e n t with MMS. ? , u n t r e a t e d control, 5 rain pulse: &, treated with o.o 4 % MMS, liltered and pulse labelled for to rain. Fig. 6. Alkaline sucrose sedimentation of I)NA synthesized by strain WP2 p o l A ~ after t r e a t m e n t with o . t ° o dichlorvos. : , u n t r e a t e d control, 5 min pulse; II, treated with n.10i, dichlorvos for 60 rain, filtered and pulse labelled for t o min.
Effect of M M S and dichlorvos on survival and molecular ~e,e.ight o[ I ) N A i~l cullur~;d Chinese hamster cells S u r v i v a l curves for MMS and dichlorvos are shown in lqg. 7. On a molar basis MMS and dichlorvos were a b o u t e q u a l l y toxic, suggesting t h a t the lethal e v e n t max, correlate with a l k y l a t i o n of protein, r a t h e r t h a n DNA. Alkaline sucrose s e d i m e n t a t i o n profiles are shown in Fig. 8. All t r e a t m e n t s received a dose of 1.8 k r a d y - i r r a d i a t i o n to p r e v e n t e n t a n g l e m e n t effects ~3. A reduction in molecular weight could be d e t e c t e d with 0.o25% MMS, a result c o m p a r a b l e to t h a t with the polA strain of E. coli. W i t h dichlorvos, a reduction in molecular weight was only observed when the c o n c e n t r a t i o n reached 0.2%. Hence dichlorvos caused nmch less " s t r a n d b r e a k a g e " in Chinese h a m s t e r cells t h a n in the polA strain of E. coli, a result more consistent with its known a l k y l a t i n g abilit3na. The reduction in molecular weight with MMS which we observe on the g r a d i e n t s m a y represent s t r a n d breaks or alkali labile lesions within the cell.
Effects of I A A on D N A A l t h o u g h on a m o l a r basis dichlorvos a l k y l a t e d D N A I5-fold less efficiently t h a n MMS 12, it is only 2-fold less p o t e n t a s t r a n d - b r e a k i n g agent in strain W P 6 7 uvrA polA. Moreover it induces chromosomal " c a t a s t r o p h e " at doses where MMS merely produces s t r a n d breaks (Figs. I a n d 2). Since the a l k y l a t i n g a n d s t r a n d - b r e a k i n g effects of dichlorvos on D N A do not correlate, it was suggested to us (P. D. LAWLE¥, pers. comm.) t h a t IAA, an a l k y l a t i n g agent which does not a t t a c k D N A to a measurable e x t e n t m i g h t produce similar effects to dichlorvos.
DICHLORVOS AND M M S INDUCED D N A i
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Top
Relotive sedimentQtion (°Io)
Fig. 7. S u r v i v a l of Chinese h a m s t e r cells (V79-4) following t r e a t m e n t for 3 ° m i n w i t h different c o n c e n t r a t i o n s of dichlorvos a n d MMS. O, MMS; A, dichlorvos. Fig. 8. Alkaline sucrose s e d i m e n t a t i o n of D N A of Chinese h a m s t e r cells (V79-4) t r e a t e d w i t h MMS a n d dichlorvos. ©, u n t r e a t e d control; A, t r e a t e d w i t h 0.2% dichlorvos for 60 rain; &, t r e a t e d w i t h 0.025% MMS for 6o min. All s a m p l e s received 1.8 k r a d 7-radiation.
Fig. 9 shows that IAA indeed caused a dramatic reduction in the molecular weight of DNA in strain WP67 uvrA polA. At low doses (0.0006 0.006%) the effect was akin to the chromosomal "catastrophe" of dichlorvos. It differed in that it occurred specifically in a polA strain, and the DNA was actually rendered acid soluble. At higher doses (0.06-0.2%) this loss of material did not occur. Instead, random strand breakage occurred similar to that found after low dose dichlorvos treatment of a polA strain. This seems to be the unusual case of an agent which causes less DNA breakdown at higher doses. Treatment with any of these levels of IAA did not affect the molecular weight of DNA in strain WP2 and produced little effect on the DNA in Chinese hamster cells (data not shown).
Effects of IAA on survival of E. coli Fig. IO shows that strains WP2, WP2 uvrA and CM56I exrA were fairly resistant to up to 0.06% IAA. Strain W P i o o was somewhat more sensitive showing greater loss of viability at 0.02% IAA. However, in keeping with the gradient experiments, strain WP67 uvrA polA was sensitive to 0.002% IAA. About 90% of the cells were killed, but there appeared to be a fraction of cells resistant up to substantially higher doses. Thus although IAA does not alkylate DNA measurably it is revealed as a DNA damaging agent from both the gradient and survival experiments with strain WP67
uvrA polA.
374
51.
I
i
b
20,
HI I,. (;RLCEN cl al.
i
lOC
I
8
E
a) .> > c
"o
Bottom
8() 610 z~O 2'0 Relative sedimentation (°/o)
Top
1
2b
do
2oo
6oo
2ooo
Iodoclcetamide con cent r'ation (Jag/m I)
Fig. 9. A l k a l i n e sucrose s e d i m e n t a t i o n of I)NA of s t r a i n W P 6 7 uvrA poL4 t r e a t e d w i t h i odoa c e t amid e. ,,, u n t r e a t e d c o n t r o l ; &, t r e a t e d w i t h 6 fig/nil for 6o m i n ; A, t r e a t e d w i t h 2o F g / m l fol6o rain; O, t r e a t e d w i t h 6 0 o / t g / m l for 6o rain. Fig. io. S u r v i v a l of s t r a i n W P 2 and its [ : V - s e n s i t i v e d e r i v a t i v e s a f t e r t r e a t m e n t for 6o m m w i t h different c o n c e n t r a t i o n s of i o d o a c e t a m i d e . O, W P 2 ; , , "vVP2 z~vrA; &, CM56I c.rrA; . \~:Ploo t¢vrA recA ; ~, \VP67 ~vr.4 poL4.
I)ISCUSSION
~),pes Q( damage The preceding e x p e r i m e n t s enable us to distinguish three different classes of d a m a g e caused b y MMS a n d two b y dichlorvos. B o t h agents produce a t y p e of s t r a n d b r e a k which is r a p i d l y r e p a i r e d b y a process involving D N A p o l y m e r a s e I, dichlorw~s a b o u t half as efficiently as MMS on a molar basis. Similar b r e a k a g e is seen with Xr a y s 2s. T r e a t m e n t with high c o n c e n t r a t i o n s of MMS, b u t not dichlorw)s, produces at/p a r e n t s t r a n d b r e a k s in the D N A of a polA + strain. T r e a t m e n t with high concentrations of dichlorvos, b u t not MMS, causes a drastic reduction of molecular weight of D N A a n d this a p p e a r s to correlate well with l e t h a l i t y . Finally, as with UV i r r a d i a t i o n ~" D N A synthesized following t r e a t m e n t with MMS is of low molecular weight, though t r e a t m e n t with dichlorw~s causes no more t h a n a small reduction in molecular weight of n e w l y - s y n t h e s i z e d D N A . These results, t o g e t h e r with o t h e r r e l e v a n t d a t a are summ a r i z e d in Table I. I t is not clear how these t y p e s of d a m a g e arise from a l k y l a t i o n of D N A and various m e c h a n i s m s could be proposed. E n z y m a t i c a t t a c k m i g h t be t h e more likely source of D N A p o l y m e r a s e I r e p a i r a b l e breaks, since a 3' O H end m u s t be formed. P e r h a p s the a l k y l a t i o n excision s y s t e m deduced to exist b y LaWLE¥ AXD ORR n is involved. The m a j o r a l k v l a t i o n p r o d u c t of MMS and dichlorvos, 7-methyl guanine it could decompose to give apurinic sites, which would be seen as alkali labile bonds in p a r e n t a l D N A a n d which couM cause discontinuities in newly-synthesized I ) N A . A l t e r n a t i v e l y discontinuities could be opposite a p a r t i c u l a r t y p e of a l k y l a t e d base.
D*ICHLORVOS A N D M M S
INDUCED
DNA
STRAND BREAKS
375
TABLE I SUMMARY OF THE EFFECTS OF MMS AND DICHLORVOS DISCUSSED IN THIS PAPER (BASED ON EQUIMOLAR CONCENTRATIONS). THE RELATIVE E F F I C I E N C I E S ARE VERY APPROXIMATE SINCE THEY ARE DOSE DEPENDENT
Property
Relative efficiency MMS
Methylation of DNAa Methylation of protein a Mutagenicity in WP2 polA +b Mutagenicity in WP67 uvrA polA b Lethality in WP2 polA +b Lethality in WP67 uvrA polA b Strand breaks in WP67 Strand breaks in WP2 "Catastrophe" Discontinuities in newlysynthesized DNA Strand breaks in Chinese hamster DNA Lethality in Chinese hamster cells
Dichlcrvos
15
i
I IO
I I
3° i 2 2
2 4 I I
+ o
o +
+++ IO I
I I
a Ref. 12. b Ref. 4. WALKER AND EWART 2~ have suggested t h a t MMS induced strand breaks in mouse Lcells are due to alkali labile phosphotriesters. An intriguing possibility from the present results is t h a t the major effects of dichlorvos and IAA on D N A are not due to alkylation of the D N A at all. Indirect effects on D N A
It is apparent from our results t h a t the relative efficiency of dichlorvos compared to MMS is greater as a D N A strand-breaking agent than as a D N A alkylating agent (see Table I). Furthermore IAA, which does not alkylate D N A to a measurable extent 1° can also cause D N A breakdown. These agents may, of course, react with D N A to give an unidentified product which is responsible for strand breakage and chromosomal " c a t a s t r o p h e " ; dichlorvos, for instance is a dimethylphosphorylating agenW. Alternatively the major effect of dichlorvos and I A A m a y be to modify enzymes and controlling elements in the cell by alkylation, so t h a t the D N A becomes subject to r a m p a n t nuclease activity. Though this hypothesis is strictly speculative it explains more readily dichlorvos " c a t a s t r o p h e " and a n u m b e r of other observations. We postulate t h a t witl-, increasing times of treatment this hypothetical nuclease is released in individual cells and rapidly degrades the DNA. Thus at intermediate times some cells have highly degraded DNA, others are unaffected (see Fig. 3). With IAA, the effects at low doses would be the same as with dichlorvos. At high doses we suggest that the suicidal nuclease activity itself becomes inhibited, so t h a t the r a m p a n t degradation is prevented (Fig. 9). It is evident (Figs. 9 and IO) that a fraction (about lO%) of the polA population is resistant to this nuclease activity. The strand breakage in the polA strain following treatment with dichlorvos and high levels of IAA might be caused by modification of another enzyme, perhaps an endonuclease, leading to incisions in normal DNA. Bacterial mutagenesis
The mutagenicity of MMS and dichlorvos in bacteria seems to correlate with the
376
M.H.L.
GI~EEN t~l ~t[.
a b i l i t y of these substances to a l k y l a t e D N A I2, r a t h e r t h a n their a b i l i t y to produce either D N A p o l y m e r a s e I repairable s t r a n d breaks or c h r o m o s o m a l " c a t a s t r o p h e " . Since b o t h agents cause mutagenesis b y misrepairL it would seem reasonable to hope t h a t the lesions subject to misrepair might show up under a p p r o p r i a t e conditions as physicochem.ical d a m a g e to D N A , either as direct s t r a n d b r e a k s (cf. 7-irradiationS, ~") or as discontinuities in n e w l y - s y n t h e s i z e d D N A (@ UV irradiationI~). In fact MMS produces b o t h classes of damage. P r o v i d e d both are subject to equally inaccurate repair the " s t r a n d b r e a k s " in p a r e n t a l D N A in a polA ~ strain will be of less i m p o r t a n c e in m u t a g e n e s i s t h a n discontinuities in newly-synthesized D N A , since such " s t r a n d b r e a k s " are fewer a n d s u b j e c t to less repair. Discontinuities in newly-synthesized D N A could also be responsible for dichlorw}s nmtagenesis, b u t we would b a r e l y be able to d e t e c t t h e m on our g r a d i e n t s at ~.~15 lower yield. M a m m a l i a n mutagencsis In Chinese h a m s t e r cells, our d a t a show t h a t in the comparison between MMS a n d dichlorvos, s u r v i v a l correlates with the e x t e n t to which the agents a l k v l a t e protein 12. D N A s t r a n d b r e a k a g e on the other hand, correlates more with the e x t e n t of a l k y l a t i o n of D N A . It is not clear w h e t h e r the breaks observed on the g r a d i e n t exist as real or p o t e n t i a l s t r a n d breaks in the cell. As far as m u t a t i o n is concerned, MMS has been d e m o n s t r a t e d to act as a m u t a g e n for these Chinese h a m s t e r cells". We are aware ot no published d a t a cn dichlorw)s. Using dichlorw~s, TURNBULI~ (pers. comm.) has been unable to induce m u t a t i o n to 8-azaguanine resistance in our Chinese h a m s t e r cells u n d e r conditions where MMS was mutagenic. If, however, mutagenesis in m a m m a l i a n cells were to be correlated with the overall a l k y l a t i o n of D N A , dichlorvos might be e x p e c t e d to be a v e r y weak m u t a g e n , b a r e l y d e t e c t a b l e b y c u r r e n t tests. ACKNOWLEDGEMENTS
We would like to t h a n k Dr. P. D. LAWLEY (Pollard's W o o d Research Station, Bucks.) Mr. B. J. DEAN (Tunstall L a b o r a t o r y , Shell Research, Sittingbourne, Kent) a n d Dr. B. A. BRIDGES for v a l u a b l e advice a n d discussion. Mrs. S. STEVENS a n d Miss W. M U R I L gave e x p e r t technical assistance and Dr. W. J. H. GRAY c o l l a b o r a t e d in p r e l i m i n a r y e x p e r i m e n t s . W e t h a n k Dr. M. G. ORMEROD who devised the calculation given in the APPENDIX, and Dr. P. D. LAWLEY for permission to quote results before publication. REFERENCES I ARLETT, C. F.,
Mutation testing with cultured mamnlalian cells, Lab. Practice, 2J (1972) 420
424 •
M., AND N. SYMONDS,Radiation sensitive mutants of T4D. I.T4y: a new radiation sensitive mutant; effect of the mutation on radiation survival, growth and recombination, iViutation Res., 8 (1969) 431 439. 3 BRIDGES, B. A., AND R. P. MOTTERSHEAD, Gamma ray mutagenesis in a straiu of Escherichia coli deficient in DNA polynlerase I, Heredity, 29 (1972) 2o3-211. 4 BRIDGES, B. A., R. P. MOTTERSHEAD, M. H. L. (;REEN AND W. J. H. GRAY, M u t a g e n i c i t y of 2 BOYLE, J.
d i c h l o r v o s a n d m e t h y l m e t h a n e s u l p h o n a t e for Escherichia coli "WP2 a n d some d e r i v a t i v e s deficient in D N A repair, 3 l u t a t i o n Res., 19 (1973) 295 303 • 5 BUHL, S. N., AND J. D. REGAN, D N A r e p l i c a t i o n in h u m a n cells t r e a t e d w i t h n l e t h v l n l e t h a n c s u l p h o n a t e , M u t a t i o n Res., 18 (1973) 191-197.
DICHLORVOS AND MMS INDUCED DNA STRAND BREAKS
377
6 CHU, E. H. Y., AND H. V. MALLING, M a m m a l i a n cell genetics, II. Chemical i n d u c t i o n of specific locus m u t a t i o n s in Chinese h a m s t e r cells, Proc. Natl. Acid. Sci. (U.S.), 61 (1968) 13o6-1312. 7 F o x , B. W., AND M. FOX, S e n s i t i v i t y of t h e n e w l y - s y n t h e s i z e d a n d t e m p l a t e D N A of l y m p h o m a cells to d a m a g e b y MMS a n d t h e n a t u r e of associated " r e p a i r " processes Mutation Res. 8 (1969) 629-638. 8 GREEN, M. H. L., W. J. H. GRAY, S. G. SEDGWICK AND n . A. BRIDGES, R e p a i r of D N A d a m a g e p r o d u c e d b y g a m m a - r a d i a t i o n in Escherichia coli K- 12 a n d a r a d i a t i o n sensitive exrA d e r i v a t i v e d u r i n g inhibition of p r o t e i n s y n t h e s i s a n d n o r m a l D N A replication b y chloramphenicol, J. Sen. Microbiol., 77 (1973) 99-1o8. 9 KAPP, D. S., AND K. C. SMITH, R e p a i r of r a d i a t i o n - i n d u c e d d a m a g e in Escherichia cull, II Effect of rec a n d uvr m u t a t i o n s on radiosensitivity, a n d repair of X - r a y i n d u c e d single s t r a n d breaks in deoxyribonucleic acid, J. Bacteriol., lO 3 (197 o) 49-64. IO LAWLEY, P. D., AND P. BROOKES, C y t o t o x i c i t y of a l k y l a t i n g a g e n t s t o w a r d s sensitive a n d res i s t a n t s t r a i n s of E. cull in relation to e x t e n t a n d m o d e of a l k y l a t i o n of cellular m a c r o m o l e c u l e s a n d repair of a l k y l a t i o n lesions in D N A , Biochem. J., lO9 (1968) 433-447I I LAWLEY, P. D., AND D. J. ORR, Specific excision of m e t h y l a t i o n p r o d u c t s f r o m D N A of E. coli t r e a t e d w i t h N-methyl-N'-nitro-N-nitrosoguanidine, Chem.-Biol. Interactions, 2 ( 197 o) 154-157. 12 LAWLEY, P. O., S. A. SHAH AND D. J. ORR, M e t h y l a t i o n of nucleic acids b y 2-2 dichlorovinyl d i m e t h y l p h o s p h a t e , Chem.-Biol. Interactions, 8 (1974) 171-182. 13 LEHMANN, A. R., P o s t r e p l i c a t i o n repair of D N A in ultraviolet~irradiated m a m m a l i a n cells, J. Mol. Biol., 66 (1972) 319-337 . 14 LEHMANN, A. R., AND M. G. ORMEROD, An a r t e f a c t in t h e m e a s u r e m e n t of t h e molecular weight of pulse labelled D N A , Nature, 221 (1969) lO53-1o56. 15 LOFROTH, G., C. KIM AND S. HUSSAIN, A l k y l a t i n g p r o p e r t y of 2-2 Dichlorovinyl D i m e t h y l P h o s p h a t e : a d i s r e g a r d e d h a z a r d , E M S Newsletter, 2 (1969) 21-27. 16 MCGRATH, R. A., AND R. W. WILLIAMS, R e c o n s t r u c t i o n in vivo of irradiated Escherichia cull deoxyribonucleic acid; t h e rejoining of b r o k e n pieces, Nature, 212 (1966) 534-535. 17 OKAZAKI, R., M. ARISAWA AND A. SUGINO, Slow j o i n i n g of n e w l y replicated D N A c h a i n s in D N A p o l y m e r a s e 1-deficient Escherichia cull m u t a n t s , Proc. Natl. Acad. Sci. (U.S.), 68 (1971) 2954-2957. 18 PRAKASH, L., AND B. STRAUSS, R e p a i r of a l k y l a t i o n d a m a g e : Stability of m e t h y l g r o u p s in Bacillus subtilis t r e a t e d w i t h m e t h y l m e t h a n e s u l p h o n a t e , J. Bacteriol., lO2 (197 o) 76o-766. 19 RuPP, W. D., AND P. HOWARD-FLANDERS, D i s c o n t i n u i t i e s in t h e D N A s y n t h e s i z e d in an excision defective s t r a i n of Escherichia cull following u l t r a v i o l e t irradiation, J. Mol. Biol., 31 (1968) 291-3o4 . 20 SEDGWlCK, S. G., AND B. A. BRIDGES, Effect of p h o t o r e a c t i v a t i o n on t h e filling of g a p s in D N A s y n t h e s i z e d after e x p o s u r e of Escherichia coli to u l t r a v i o l e t light, J. Bacteriol., 117 (1974) lO77-1o81. 21 SETLOW, R. B., J. D. REGAN, J. GERMAN AND W. L. CARRIER, E v i d e n c e t h a t Xeroderma pigmentosum cells do n o t p e r f o r m t h e first step in t h e repair of u l t r a v i o l e t d a m a g e to their D N A , Proc. Natl. Acid. Sci. (U.S.), 64 (1969) lO35-1o41. 22 SINCLAIR, W. K., AND R. A. MORTON, X - r a y a n d u l t r a v i o l e t s e n s i t i v i t y of s y n c h r o n i s e d Chinese h a m s t e r cells a t v a r i o u s stages of t h e cell cycle, Biophys. J . , 5 (1965) 1-25. 23 STRAUSS, B. S., AND R. WAHL, T h e presence of baeaks in t h e D e o x y r i b o n u c l e i c acid of Bacillus subtilis t r e a t e d in vivo w i t h t h e a l k y l a t i n g a g e n t m e t h y l m e t h a n e s u l p h o n a t e , Biochim. Biophys. Acta, 80 (1964) 116-126. 24 STUDIER, F. W., S e d i m e n t a t i o n s t u d i e s of t h e size a n d s h a p e of D N A , J. Mol. Biol., i i (1965)373. 25 TOWN, C. D., K. C. SMITH AND S . S. KAPLAN, D N A p o l y m e r a s e required for t h e rapid rejoining of X - r a y i n d u c e d D N A s t r a n d b r e a k s in vivo, Science, 172 (1971) 851-853. 26 WALKER, I. G., AND D. F. EWART, T h e n a t u r e of single s t r a n d b r e a k s following t r e a t m e n t of L-cells w i t h m e t h y l a t i n g agents, Mutation Res., ~9 (1973) 331-341. 27 BEDFORD, C. T., AND J. ROBINSON, T h e a l k y l a t i n g properties of o r g a n o p h o s p h a t e s Xenobiotica, 2 (1972) 3o7-337 • APPENDIX
C a l c u l a t i o n o f n u m b e r - a v e r a g e molecular weights The number-average
Mn =
m o l e c u l a r w e i g h t of a p o p u l a t i o n
nMdM o
ndM •
o
of m o l e c u l e s is d e f i n e d a s
(z)
378
M.H.L. GREEN et al.
where n is the n u m b e r of molecules of molecular weight between M and M + d M . For a r a n d o m distribution of molecules (produced by the r a n d o m introduction of more than about five breaks per molecule into an initial population of molecules) n ~ A exp(--M/M~) where A is a constant (adapted from CHARLESBy1). Let us consider the apparent number-average molecular weight M'n obtained by integrating equation (z) from a finite lower limit M1, for a r a n d o m distribution. M,' =
M/Mn exp(-1
Mn)d
ex
--M/Mn)dM
M1
: M~ + M 1
(2)
i.e., to obtain the true value Mn, a correction factor equal to M1 must be subtracted from M'n. Applying the above to a discrete radioactivity distribution from the fractions in a sucrose gradient, equation (5) gives oo
oo
Mn = Z C , / Z C~/M,, i=o
(3)
i=o
where Ct is the radioactivity in a fraction of average molecular weight M, (assuming t h a t the molecules are uniformly labelled). Direct calculation of Mn from a radioactivity distribution from a sucrose gradient b y this summation is extremely inaccurate. The summation is strongly influenced b y fractions at the top of the gradient ; these are often contaminated with a great deal of spurious material (e.g. see Fig. 2). This causes the summation to be grossly in error. For a r a n d o m distribution of molecules, Mn can be accurately determined by a graphical method, which also tests the randomness of the distribution ~,~. Alternatively Mn m a y be calculated b y carrying out the above summation from a finite lower limit and applying the correction factor from equation (2). The graphical method demonstrated t h a t the radioactivity profiles obtained in our experiments were not in fact r a n d o m and it could not therefore be used to calculate Mn. Equation (2) is also only strictly valid for a r a n d o m distribution of molecules. However, the profiles we , were considering did not deviate very much from randomness below the top 4-6 fractions so t h a t errors in using the correction factor M1 should only produce a second-order error in the value of M,. This method has therefore been used for calculation of Mn although it is appreciated t h a t the calculations are not rigorously valid. REFERENCES (Appendix) A., Molecular weight changes in the degradation of long-chain polymers, Proc. Roy. Soc. (London), Ser. A, 224 (1954) 12o. 2 DEAN, C. J., M. G. ORMEROD,R. W. SERRIANIAND P. ALEXANDER,DNA strand breakage in cells irradiated with X-rays, Nature, 222 (1969) lO42. 3 LEHMANN, A. R., AND M. G. ORMEROD, The replication of DNA in murine lymphoma cells (L 5178Y) I rate of replication, Biochim. Biophys. Acta, 204 (197o) 128-143. I CHARLESBY,
365
DNA STRAND B R E A K A G E CAUSED BY DICHLORVOS, M E T H Y L M E T H A N E S U L P H O N A T E AND IODOACETAMIDE IN ESCHERICHIA COLI AND C U L T U R E D C H I N E S E HAMSTER CELLS
M. H. L. GREEN, A. S. C. MEDCALF, C. F. ARLETT, S. A. HARCOURT AND A. R. LEHMANN
MRC Cell Mutation Unit, University of Sussex, Falmer, Brighton, BNr 90,G (Great Britain) (Received February 5th, 1974)
SUMMARY
The DNA damaging properties of dichlorvos (2,2 dichlorovinyl dimethyl phosphate), methyl methanesulphonate (MMS) and iodoacetamide (IAA) have been studied, using alkaline sucrose sedimentation. In a strain of E. coli deficient in DNA polymerase I (polA) both dichlorvos and MMS caused random strand breakage, MMS being about twice as efficient as dichlorvos on a molar basis. In pol + bacteria, DNA strand breaks or alkali labile bonds were detected following treatment with roughly five-fold higher concentrations of MMS but at similar high concentrations of dichlorvos there was an all or none breakdown of DNA molecules to fragments of very low molecular weight which correlated well with lethality. DNA synthesized after treatment of pol÷ and polA bacteria with MMS was of low molecular weight, indicating the presence of discontinuities. With dichlorvos, the effect was much smaller. Apparent all-or-none DNA breakdown was also found when the polA strain of E. coli was treated with low concentrations of iodoacetamide, an agent that does not detectably alkylate DNA. At higher concentrations the breakdown was suppressed and random strand breakage occurred instead. These effects did not occur with pol÷ bacteria and correlated well with the greater sensitivity to iodoacetamide of the polA strain in survival experiments. We suggest that the major DNA damage resulting from treatment with iodoacetamide and dichlorvos arises indirectly through alkylation of other cellular constituents and consequent uncontrolled nuclease attack on the DNA. Discontinuities in newly synthesized DNA and mutagenesis following dichlorvos treatment, however, presumably result from direct alkylation of DNA. Strand breakage caused by dichlorvos and MMS in Chinese hamster cells tended to correlate with the extent to which these agents alkylate DNA, but survival tended to correlate with the alkylation of protein.
Abbreviations: dichlorvos, 2,2 dichlorovinyl dimethyl phosphate; IAA, iodoacetamide; MMS, methyl methanesulphonate.
366
M.H.L.
GREEN cl ol.
INTRODUCTION
The insecticide dichh;rvos has been shown to alkylate DNA ~ and may be a potential environmental nmtagen. With regard to the types of alkylations produced in DNA dichlorvos resembles MMS more closely than other common alkylating agents although dichlorvos has the additional capacity to dimethylphosphorylate protein2L On a molar basis it is about as effective as MMS in alkylating protein but about 15-fold less potent in alkylating DNA ~2. BRI1)GES et al. ~ have compared the lethal and mutagenie effects of dichtorw~s with those of MMS in a series of radiati(m-sensitive strains of Escherichia coll. ExrA, RecA and PolA strains of E. coli show 4 increased sensitivity t o both agents. Mutagenesis with both dichhnvos and MMS occurs 1)\, misret)air (i.e. ExrA or RecA strains are not mutated). There is, however, nmch less diffeience in survival between resistant and sensitive strain, s with (tichlorvos than with MMS, an(1 dichlmvos is a nmch weaker mutagen L lake MMS'S,v,~s,'za,z6 dichlorvos causes DNA strand breakage a and we wondered if it were misrepair of such straud breaks that made these agents mutagenic. If so, the differences in nmtagenicity between dichlorvos and MMS might relate to differences in the type and number of strand breaks. In this paper we show that (tichh)rw~s and MMS do differ in the type of strand breakage they cause. We also find that dichloi vos appears to cause more damage to I)NA in E. col," than can be accounted for by direct alkylation. We have consi(lered the possibility that such damage may occur indirectly, by alkylation of other cellular constituents leading to enzymatic attack on DNA, and, in this connection we have shown that IAA, an agent that does not alkylate I)NA to a measurable extent ~°, can also cause DNA damage. Finally, we have extended our study' to include the lethal and strand-breaking effects of dichlorvos in cultured Chinese hamster cells. MATERIALS AND METHODS
Escherichia coli Strains. WP2 (UV resistant), WP67 uvrA polA, WP2 uvrA, CM56I exrA and W P I o o uvrA recA have been described previously and are approximately isogenicL We are indebted to Dr. E. M. WITKIN for supplying strains WP67 and WPIoo. Media. Growth medium was M9 salts supplemented with : Casamino acids 1% ; glucose o.5}/o • tri-sodimn citrate I mg/ml and tryptophan 25 Ftg/ml. Citrate was required for the growth nf strain WP67. Buffer was BOYLE AND SYMONDS2 phosphate buffer. Viabilities were determined on nutrient agar (Oxoid Nutrient Broth No. 2 solidified with 1.5°/i~ New Zealand Agar). Reagents. [all]methyl thymidine, 22 ('i/mmole, the Radiochemical Centre, Amersham, Bucks. Dichlorvos was kindly given to us by Mr. B. J. DEAN, Tunstall Laboratory, Shell Research, Sittingbourne, Kent. MMS, Ralph N. Emmanuel, Wembley, Middlesex. IAA, Sigma Ltd., London. Sarkosyl, Geigy Ltd., Manchester. Dulbecco's troffer A, Oxoid, London. Protocol. Overnight cultures of E. coli were diluted into glowth medium containing [aHlmethyl thymidine (5° ~Ci/ml; 0. 5/~g/ml) and IOO/~g/ml deoxyadenosine. They were incubated for about 2. 5 h, filtered and the cells resuspended in buffer. A known volume was added to a bottle containing an ethanolic solution of dichlorvos or
DICHLORVOS AND M M S INDUCED D N A STRAND BREAKS
367
an aqueous solution of IAA to give an appropriate final concentration of the agent. Stocks of dichlorvos and MMS were freshly made up and diluted in analar ethanol. The final concentration of ethanol during treatment did not exceed 20/0. IAA was freshly made up in buffer. Except where otherwise indicated, treatment was for 60 rain at 37 ° with shaking. Following treatment cells were layered directly on alkaline sucrose gradients. When repair cf initial damage was examined cells were filtered, resuspended in growth medium, incubated further at 37 ° and samples taken at intervals. To examine the molecular weight of newly-synthesized DNA, unlabelled cultures were treated, filtered and resuspended in growth medium containing 50/~Ci/ml ~3H]methyl thymidine (specific activity 22 Ci/mmole) and ioo/~g/ml deoxyadenosine. After IO rain they were filtered again and resuspended in growth medium without label. Samples were layered on alkaline sucrose gradients at intervals. It was necessary to halve the culture volume at each filtration to compensate for losses of cells on the filter. In survival experiments cells were treated with IAA in similar fashion to gradient experiments, diluted at least ioo-fold and plated on nutrient agar.
Chinese hamster The Chinese hamster cell line V79-4 was routinely maintained in Eagle's Minimal Essential Medium (MEM) with 15% foetal calf serum (Flow Laboratories) and was used during passages 7 ° to 92 . Our tissue culture techniques and materials have been described in detail elsewhere 1. Eor investigation of colony-forming ability cells in the exponential growth phase were trypsinized (0.025% for io rain) from monolayer culture and after centrifugation and resuspension in culture medium a range of dilutions was dispensed into 5 cm petri dishes (Sterilin Ltd) in 5 ml medium. After 4 h incubation to allow the cells to become attached to the surface and recommence growth, but before any increase in cellular multiplicity2L the medium was removed. Two ml of Dulbecco's buffer A was added, containing an appropriate concentration of dichlorvos or MMS. Stocks of lO% MMS and dichlorvos in ethanol were freshly prepared and diluted in buffer. The final concentration of ethanol did not exceed 0.8% in any treatment and this level of ethanol was also added to the controls. Treatment was for 30 rain at 37 °, after which the buffer was removed and replaced with 5 ml fresh medium. The treated plates were incubated for 8 days before staining with I ~o methylene blue. The criterion for survival was the production of a clone containing more than 50 cells. For the strand breakage studies, on day i cells were dispensed at I × lO 5 cells per 5 cm petri dish in 5 ml medium during the morning. At 5 p.m. the medium was supplemented with E3Hlmethyl thymidine (5 #Ci/ml; I/~M) and the plates incubated overnight. On day 2 the culture medium was removed and replaced with 2 ml Dulbecco's buffer A. Fresh ethanolic solutions of dichlorvos and MMS were added to give an appropriate final concentration. The level of ethanol did not exceed 2% in any treatment. Treatment was for 60 min at 37 °, following which the buffer was removed and replaced with 0.3 ml of Dulbecco's buffer A solution containing 0.2 g/1 E D T A ~1. The cells were scraped off with a piece of silicon rubber.
.x.L H. L. {;REEN el al.
368
Alkaline sucrose sedimentation E. coli. Samples of o.o8 ml or less were lysed in a l a y e r of o. i ml S a r k o s y l lysis m i x t u r e (Sarkosyl, I }; ; N a O H , 0. 5 M; NaC1, o.2 M ; E D T A o.oi M) on a linear g r a d i e n t of 5-2O~o (w/v) sucrose in 0.2 N N a O H , 0.5 N NaC1 a n d o.ooi M E D T A . The c o n c e n t r a t i o n of o.oi M E D T A in the lysis m i x t u r e , r a t h e r t h a n o.oo~ :11 as used p r e v i o u s l y 8 was found to give b e t t e r results. The g r a d i e n t s were spun at 27 00o r e v . / m i n for 9 ° rain in a Spinco S W 5 o . I rotor. F r a c t i o n s were collected on strips of W h a t m a n 3 MM paper, washed and c o u n t e d as described previously ". Chinese hamster. Before layering, sanlples received a dose of I.S k r a d v-irradiation in order to reduce the molecular weight sufficiently to prevent the s e d i m e n t a t i o n p r o b l e m s which occur with v e r y high m o l e c u l a r weight D N A ~3. Samples of 0.o 5 ml or less (to give less t h a n IO 4 cells per gradient) were lvsed in a lavcr of o.I ml of 2 °,/ o . s o d i u m d o d e c y l s u l p h a t e and o.02 M E D T A above a linear g r a d i e n t of a 5 - 2 o % (w/v) sucrose in o.I M NaC1 and o.I M N a O H . G r a d i e n t s were spun at 38ooo r e v . / m i n for 7 ° min a n d fractions were collected as with E. coli. Similar results were o b t a i n e d o m i t t i n g the ;,-irradiation a n d using a CsCl-sucrose shelf to collect the high molecular weight m a t e r i a h
Calculation of induced stra~zd-break freque~wies It Mo a n d M,~ are the n u m b e r average molecular-weights of the u n t r e a t e d and t r e a t e d p o p u l a t i o n s of D N A molecules, the n u m b e r of s t r a n d breaks p r o d u c e d per average u n t r e a t e d molecule is equal to
Mo/M,,-- I Molecular weights (M) of each fraction were calculated using a relationship derived from STUDIER2L M ~ D 2.5 where D is the distance s e d i m e n t e d b y molecules in t h a t fraction. Values of Mo a n d M,~ were d e t e r m i n e d from e q u a t i o n (3) in the A p p e n d i x : .14n
=
:]In'
-- M 1
where M ( is the n u m b e r - - a v e r a g e molecular weight of those molecules which had s e d i m e n t e d further t h a n the sixth fraction from the t o p of t h e gradient, a n d 3 [ t ( = 8 × IO 6) is the n o m i n a l molecular weight of molecules in the sixth fraction. Since we were only i n t e r e s t e d in relative efficiencies of s t r a n d breakage, no a t t e m p t was m a d e to c a l i b r a t e the g r a d i e n t s accurately. Mo was a p p r o x i m a t e l y its n o m i n a l value I x IO ~. RESULTS
Strand breakage in a polA strain Profiles of s t r a n d b r e a k a g e caused b y dichlorvos a n d MMS in strain W P 0 7
uvrA polA have been published p r e v i o u s l y 4. I n d u c e d s t r a n d b r e a k frequencies from a series of similar e x p e r i m e n t s were calculated, a v e r a g e d a n d p l o t t e d in Fig. I. The induction of s t r a n d b r e a k s b y MMS in strain W P 6 7 uvrA polA seemed to give a nonlinear dose-response. Nevertheless, on a m o l a r basis, dichlorvos was roughly half as efficient as MMS in inducing s t r a n d breaks in a polA strain. S t r a n d breaks could not be d e t e c t e d at low doses of MMS and dichlorw>s in a polA + strain, suggesting t h a t the b r e a k s o b s e r v e d at these doses in strain W P 6 7
DICHLORVOS
AND
MMS
INDUCED
DNA
STRAND
369
BREAKS
would, in the polA + strain, be rapidly repaired by DNA polymerase I, a situation similar to that found with X-rays 2~. If repair by DNA polymerase I is error freeL these breaks are unlikely to he a major cause of dichlorvos or MMS mutagenesis in a polA ÷ strain. Since DNA polymerase I is normally associated with the repolymerization step of strand rejoining it would seem likely that the lesions exist in the polA strain as actual breaks rather than alkali labile bonds. We cannot say whether such breaks are formed enzymatically or chemically.
Strand breakage by M M S in a polA + strain From Fig. I it can be seen that the number of MMS induced strand breaks was about 5-6-fold less in strain WP2 poIA + than in strain WP67 uvrA polA so that breaks were only detectable at higher doses in the former. Fig. 2 shows the result of an experiment in which strain WP2 was treated with o.I}/o MMS for 60 min, the MMS removed and incubation continued for a further 60 rain in a growth medium. Treatment with o.1% MMS caused a considerable initial reduction in the molecular weight of the DNA on the gradient which was partly but not completely restored during further incubation. Following treatment with 0.2% MMS little restoration of molecular weight occurred during subsequent incubation (data not shown). The reduction in molecular weight in Fig. 2 m a y represent actual breaks in the DNA in the cell or alkali labile lesions. 005 0.1 0.025 005
0.2 Dichlorvos(%) 01 MMS(°/o) 0.2 i
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Fig. I. C a l c u l a t e d i n d u c e d D N A s t r a n d b r e a k frequencies for s t r a i n s W P 6 7 uvrA polA a n d W P 2 polA + t r e a t e d w i t h d i c h l o r v o s a n d MMS. F r e q u e n c i e s are n o r m a l i s e d a g a i n s t t h e c o n t r o l n u m b e r a v e r a g e m o l e c u l a r w e i g h t (approx. i × i o 8 daltons). B a s e d on t h e a v e r a g e of 3 - 6 e x p e r i m e n t s . O, MMS polA ; 0, d i c h l o r v o s polA ; &, MMS polA +. Fig. 2. A l k a l i n e sucrose s e d i m e n t a t i o n of D N A of s t r a i n W P 2 polA + t r e a t e d w i t h MMS. O, unt r e a t e d c o n t r o l ; &, t r e a t e d w i t h o.i % MMS for 6o rain; e , t r e a t e d w i t h o.I % MMS for 60 min, r e s u s p e n d e d a n d i n c u b a t e d in g r o w t h m e d i u m for 60 min.
37 o
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D N A "catastrophe" caused by dichlorvos T r e a t m e n t of strain W P 2 with dichlorvos gave a result quite different from t h a t o b t a i n e d with MMS. I n s t e a d of a s t e a d y shift of the peak to lower molecular weight with increasing dose, the peak m o v e d d i r e c t l y from n o r m a l to w'r\, h)w molecular weight. R a t h e r t h a n r a n d o m s t r a n d breakage, it a p p e a r e d t h a t i n d i v i d u a l D N A molecules had d i s i n t e g r a t e d in an all-or-none fashion a n d a p p e a r e d in the low molecular weight position. This usually occurred when strain W P 2 was t r e a t e d with v e r y high c o n c e n t r a t i o n s of dichlorvos (between a b o u t (>.20/0 and o.4'~ , v/v)* and corresponded well with our previous o b s e r v a t i o n 4 of a sharp loss of viahilitv between these concentrations. Lower c o n c e n t r a t i o n s of dichlorw)s had no d e t e c t a b l e effect ()n DNA. An e x a m p l e of this c h r o m o s o m a l " c a t a s t r o p h e " is shown in l;ig. 3. Strain \\"P2 w a s t r e a t e d with 0.4% dichlorvos and samples t a k e n at o, Io, 2o and 4 ° rain. It can be seen t h a t the profile after IO rain represents two p o p u l a t i o n s r a t h e r t h a n a distribution of r a n d o m l y broken molecules; some D N A molecules were u n b r o k e n , others were s h a t t e r e d . A f t e r 20 rain most of the D N A h a d shifted from normal to low molecular weight. W e found no evidence t h a t dichlorvos i n d u c e d a t y p e of s t r a n d breakage similar to t h a t caused by MMS in strain W P 2 ; it only a p p e a r e d to induce c a t a s t r o p h i c c h r o m o s o m a l b r e a k d o w n . The low molecular weight m a t e r i a l was not repaired bu~ was further d e g r a d e d to acid-soluble m a t e r i a l when the cells were r e t u r n e d to growth nredium (data not shown).
Molecular weight of DNA .2vnthesized following treatment with M M S a~2d dichlorvos In order to e x a m i n e the size of newly-synthesized D N A , t r e a t e d and u n t r e a t e d cells were pulse-labelled with 3 H , t h y m i d i n e . To m a k e valid comparisons, the pulse times were a d j u s t e d so t h a t the a m o u n t of D N A synthesized in u n t r e a t e d an(l t r e a t e d cells was similar. This avoids labelling artefacts similar to those described by LV.HMANN A N D O R M E R O I ) 14 a n d ~ E D G W I C K AND B R I D G E S ~°. l:ollowing t r e a t m e n t with o.o4% MMS, D N A of low molecular weight was synthesized in strain W P 2 (Irig. 4) ~. The m o l e c u l a r weight increased to t h a t of u n t r e a t e d cells on further incubation. This situation seems to be similar to t h a t seen after U V - i r r a d i a t i o n ~'. P r e s u m a b l y the new D N A c o n t a i n e d discontinuities opposite some lesion in the p a r e n t a l strands, and these were s u b s e q u e n t l y filled in. I t was not possible to calculate a useful frequency of gaps induced in newlysynthesized D N A for several reasons. A direct calculation of n u m b e r average molecular weight would be worthless, because of the error i n t r o d u c e d bv spuriously s e d i m e n t i n g m a t e r i a l in the t o p few fractions. The correction described in the APPt.:N1~IX could not be e m p l o y e d , because the m a t e r i a l s e d i m e n t i n g further t h a n the t o p fractions did not even a p p r o x i m a t e to a r a n d o m b r e a k a g e profile. In the calculations for Fig. I a p p r o x i m a t e l y equal values for n u m b e r average molecular weight were o b t a i n e d with cut-off points (M1) of 0.08 × Mo, 0.44 / M,, or 1.32 / M,, which suggests * A l t h o u g h t h e s o l u b i l i t y of d i c h l o r v o s in w a t e r is o n l y a b o u t o. I (}i) (B. J. DEAN, pers. c o m m . ) wc c o n s i s t e n t l y o b t a i n i n c r e a s e s in e f f e c t of d i c h l o r v o s w i t h n o m i n a l c o n c e n t r a t i o n s as h i g h as o.2'>, o r o .4 o. .'o. \ ¥ h e n d i c h l o r v o s is a d d e d as a 4 o o s o l u t i o n in e t h a n o l , it is s e e n t o s e p a r a t e b u t n o t w h e n a d d e d as a 2 o % s o l u t i o n . T h e a d d i t i o n a l d i c h l o r v o s m i g h t d i s s o l v e p a r t l y in t h e e t h a n o l , o r p a r t l y in cell c o n s t i t u e n t s .
DICHLORVOS AND
MMS INDUCED DNA STRAND BREAKS i
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Fig. 3. Alkaline sucrose sedimentation of D N A of strain WP2 polA + treated with 0.4% dichlorvos. O, u n t r e a t e d contlol; A, treated with dichlorvos for io min; £, treated for 2o min; O, treated foz 4 ° min. Fig. 4- Alkaline sucrose sedimentation of D N A synthesized by strain W P 2 polA + after t r e a t m e n t with MMS. O, u n t r e a t e d control, IO min pulse; 4, treated with o.o5% MMS for 6o min filtered, pulse labelled for IO min, refiltered and resuspended in g r o w t h m e d i u m and sampled; e , t r e a t e d w i t h MMS, labelled, refiltered etc., incubated 60 min before sampling. The counts in the treated samples have been multiplied x 5 to allow for filtration losses.
that this method gives a good estimate of Mn. With a hyperdisperse profile, like the control in Fig. 4 the calculated number-average molecular weight would increase in an arbitrary way with the cut-off point chosen. We therefore investigated calculations which were based on the heavier fractions and which were not dependent on a control with a random breakage profile. These, however, were vitiated by another difficulty. It was evident from Fig. 4 and other experiments that some gap filling was occurring in the treated sample during the IO min pulse. This meant that the heaviest material in the treated profile did not give a true indication of the induced frequency of gaps and could not be used either. Nevertheless after a given dose of MMS the daughter strand discontinuities seemed to be greater in number than the strand breaks in parental DNA (Figs. I and 2), and seemed to be repaired more completely. With neither type of lesions did further joining occur with periods of incubation greater than 6o min. Treatment with o.o4% MMS also induced discontinuities in newly-synthesized DNA in strain WP67 uvrA polA (Fig. 5)- Though consistent the effect appears less marked because as is evident from Fig. 5 the control newly-synthesized DNA was also of low molecular weight'74 °. Again it was not possible to calculate a useful frequency for induced gaps or determine whether or not MMS caused an equal number of gaps in newly-synthesized DNA in a polA and polA + strain. Finally Fig. 6 shows that treatment with o.1% dichlorvos caused no more than a slight reduction in the molecular weight of newly-synthesized DNA in strain WP2.
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Relative sedimentation
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Fig. 5. Alkaline sucrose sedimentation of DNA synthesized by strain \VP07 u v r A p o l A after t r e a t m e n t with MMS. ? , u n t r e a t e d control, 5 rain pulse: &, treated with o.o 4 % MMS, liltered and pulse labelled for to rain. Fig. 6. Alkaline sucrose sedimentation of I)NA synthesized by strain WP2 p o l A ~ after t r e a t m e n t with o . t ° o dichlorvos. : , u n t r e a t e d control, 5 min pulse; II, treated with n.10i, dichlorvos for 60 rain, filtered and pulse labelled for t o min.
Effect of M M S and dichlorvos on survival and molecular ~e,e.ight o[ I ) N A i~l cullur~;d Chinese hamster cells S u r v i v a l curves for MMS and dichlorvos are shown in lqg. 7. On a molar basis MMS and dichlorvos were a b o u t e q u a l l y toxic, suggesting t h a t the lethal e v e n t max, correlate with a l k y l a t i o n of protein, r a t h e r t h a n DNA. Alkaline sucrose s e d i m e n t a t i o n profiles are shown in Fig. 8. All t r e a t m e n t s received a dose of 1.8 k r a d y - i r r a d i a t i o n to p r e v e n t e n t a n g l e m e n t effects ~3. A reduction in molecular weight could be d e t e c t e d with 0.o25% MMS, a result c o m p a r a b l e to t h a t with the polA strain of E. coli. W i t h dichlorvos, a reduction in molecular weight was only observed when the c o n c e n t r a t i o n reached 0.2%. Hence dichlorvos caused nmch less " s t r a n d b r e a k a g e " in Chinese h a m s t e r cells t h a n in the polA strain of E. coli, a result more consistent with its known a l k y l a t i n g abilit3na. The reduction in molecular weight with MMS which we observe on the g r a d i e n t s m a y represent s t r a n d breaks or alkali labile lesions within the cell.
Effects of I A A on D N A A l t h o u g h on a m o l a r basis dichlorvos a l k y l a t e d D N A I5-fold less efficiently t h a n MMS 12, it is only 2-fold less p o t e n t a s t r a n d - b r e a k i n g agent in strain W P 6 7 uvrA polA. Moreover it induces chromosomal " c a t a s t r o p h e " at doses where MMS merely produces s t r a n d breaks (Figs. I a n d 2). Since the a l k y l a t i n g a n d s t r a n d - b r e a k i n g effects of dichlorvos on D N A do not correlate, it was suggested to us (P. D. LAWLE¥, pers. comm.) t h a t IAA, an a l k y l a t i n g agent which does not a t t a c k D N A to a measurable e x t e n t m i g h t produce similar effects to dichlorvos.
DICHLORVOS AND M M S INDUCED D N A i
STRAND BREAKS
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/o
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Fig. 7. S u r v i v a l of Chinese h a m s t e r cells (V79-4) following t r e a t m e n t for 3 ° m i n w i t h different c o n c e n t r a t i o n s of dichlorvos a n d MMS. O, MMS; A, dichlorvos. Fig. 8. Alkaline sucrose s e d i m e n t a t i o n of D N A of Chinese h a m s t e r cells (V79-4) t r e a t e d w i t h MMS a n d dichlorvos. ©, u n t r e a t e d control; A, t r e a t e d w i t h 0.2% dichlorvos for 60 rain; &, t r e a t e d w i t h 0.025% MMS for 6o min. All s a m p l e s received 1.8 k r a d 7-radiation.
Fig. 9 shows that IAA indeed caused a dramatic reduction in the molecular weight of DNA in strain WP67 uvrA polA. At low doses (0.0006 0.006%) the effect was akin to the chromosomal "catastrophe" of dichlorvos. It differed in that it occurred specifically in a polA strain, and the DNA was actually rendered acid soluble. At higher doses (0.06-0.2%) this loss of material did not occur. Instead, random strand breakage occurred similar to that found after low dose dichlorvos treatment of a polA strain. This seems to be the unusual case of an agent which causes less DNA breakdown at higher doses. Treatment with any of these levels of IAA did not affect the molecular weight of DNA in strain WP2 and produced little effect on the DNA in Chinese hamster cells (data not shown).
Effects of IAA on survival of E. coli Fig. IO shows that strains WP2, WP2 uvrA and CM56I exrA were fairly resistant to up to 0.06% IAA. Strain W P i o o was somewhat more sensitive showing greater loss of viability at 0.02% IAA. However, in keeping with the gradient experiments, strain WP67 uvrA polA was sensitive to 0.002% IAA. About 90% of the cells were killed, but there appeared to be a fraction of cells resistant up to substantially higher doses. Thus although IAA does not alkylate DNA measurably it is revealed as a DNA damaging agent from both the gradient and survival experiments with strain WP67
uvrA polA.
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2ooo
Iodoclcetamide con cent r'ation (Jag/m I)
Fig. 9. A l k a l i n e sucrose s e d i m e n t a t i o n of I)NA of s t r a i n W P 6 7 uvrA poL4 t r e a t e d w i t h i odoa c e t amid e. ,,, u n t r e a t e d c o n t r o l ; &, t r e a t e d w i t h 6 fig/nil for 6o m i n ; A, t r e a t e d w i t h 2o F g / m l fol6o rain; O, t r e a t e d w i t h 6 0 o / t g / m l for 6o rain. Fig. io. S u r v i v a l of s t r a i n W P 2 and its [ : V - s e n s i t i v e d e r i v a t i v e s a f t e r t r e a t m e n t for 6o m m w i t h different c o n c e n t r a t i o n s of i o d o a c e t a m i d e . O, W P 2 ; , , "vVP2 z~vrA; &, CM56I c.rrA; . \~:Ploo t¢vrA recA ; ~, \VP67 ~vr.4 poL4.
I)ISCUSSION
~),pes Q( damage The preceding e x p e r i m e n t s enable us to distinguish three different classes of d a m a g e caused b y MMS a n d two b y dichlorvos. B o t h agents produce a t y p e of s t r a n d b r e a k which is r a p i d l y r e p a i r e d b y a process involving D N A p o l y m e r a s e I, dichlorw~s a b o u t half as efficiently as MMS on a molar basis. Similar b r e a k a g e is seen with Xr a y s 2s. T r e a t m e n t with high c o n c e n t r a t i o n s of MMS, b u t not dichlorw)s, produces at/p a r e n t s t r a n d b r e a k s in the D N A of a polA + strain. T r e a t m e n t with high concentrations of dichlorvos, b u t not MMS, causes a drastic reduction of molecular weight of D N A a n d this a p p e a r s to correlate well with l e t h a l i t y . Finally, as with UV i r r a d i a t i o n ~" D N A synthesized following t r e a t m e n t with MMS is of low molecular weight, though t r e a t m e n t with dichlorw~s causes no more t h a n a small reduction in molecular weight of n e w l y - s y n t h e s i z e d D N A . These results, t o g e t h e r with o t h e r r e l e v a n t d a t a are summ a r i z e d in Table I. I t is not clear how these t y p e s of d a m a g e arise from a l k y l a t i o n of D N A and various m e c h a n i s m s could be proposed. E n z y m a t i c a t t a c k m i g h t be t h e more likely source of D N A p o l y m e r a s e I r e p a i r a b l e breaks, since a 3' O H end m u s t be formed. P e r h a p s the a l k y l a t i o n excision s y s t e m deduced to exist b y LaWLE¥ AXD ORR n is involved. The m a j o r a l k v l a t i o n p r o d u c t of MMS and dichlorvos, 7-methyl guanine it could decompose to give apurinic sites, which would be seen as alkali labile bonds in p a r e n t a l D N A a n d which couM cause discontinuities in newly-synthesized I ) N A . A l t e r n a t i v e l y discontinuities could be opposite a p a r t i c u l a r t y p e of a l k y l a t e d base.
D*ICHLORVOS A N D M M S
INDUCED
DNA
STRAND BREAKS
375
TABLE I SUMMARY OF THE EFFECTS OF MMS AND DICHLORVOS DISCUSSED IN THIS PAPER (BASED ON EQUIMOLAR CONCENTRATIONS). THE RELATIVE E F F I C I E N C I E S ARE VERY APPROXIMATE SINCE THEY ARE DOSE DEPENDENT
Property
Relative efficiency MMS
Methylation of DNAa Methylation of protein a Mutagenicity in WP2 polA +b Mutagenicity in WP67 uvrA polA b Lethality in WP2 polA +b Lethality in WP67 uvrA polA b Strand breaks in WP67 Strand breaks in WP2 "Catastrophe" Discontinuities in newlysynthesized DNA Strand breaks in Chinese hamster DNA Lethality in Chinese hamster cells
Dichlcrvos
15
i
I IO
I I
3° i 2 2
2 4 I I
+ o
o +
+++ IO I
I I
a Ref. 12. b Ref. 4. WALKER AND EWART 2~ have suggested t h a t MMS induced strand breaks in mouse Lcells are due to alkali labile phosphotriesters. An intriguing possibility from the present results is t h a t the major effects of dichlorvos and IAA on D N A are not due to alkylation of the D N A at all. Indirect effects on D N A
It is apparent from our results t h a t the relative efficiency of dichlorvos compared to MMS is greater as a D N A strand-breaking agent than as a D N A alkylating agent (see Table I). Furthermore IAA, which does not alkylate D N A to a measurable extent 1° can also cause D N A breakdown. These agents may, of course, react with D N A to give an unidentified product which is responsible for strand breakage and chromosomal " c a t a s t r o p h e " ; dichlorvos, for instance is a dimethylphosphorylating agenW. Alternatively the major effect of dichlorvos and I A A m a y be to modify enzymes and controlling elements in the cell by alkylation, so t h a t the D N A becomes subject to r a m p a n t nuclease activity. Though this hypothesis is strictly speculative it explains more readily dichlorvos " c a t a s t r o p h e " and a n u m b e r of other observations. We postulate t h a t witl-, increasing times of treatment this hypothetical nuclease is released in individual cells and rapidly degrades the DNA. Thus at intermediate times some cells have highly degraded DNA, others are unaffected (see Fig. 3). With IAA, the effects at low doses would be the same as with dichlorvos. At high doses we suggest that the suicidal nuclease activity itself becomes inhibited, so t h a t the r a m p a n t degradation is prevented (Fig. 9). It is evident (Figs. 9 and IO) that a fraction (about lO%) of the polA population is resistant to this nuclease activity. The strand breakage in the polA strain following treatment with dichlorvos and high levels of IAA might be caused by modification of another enzyme, perhaps an endonuclease, leading to incisions in normal DNA. Bacterial mutagenesis
The mutagenicity of MMS and dichlorvos in bacteria seems to correlate with the
376
M.H.L.
GI~EEN t~l ~t[.
a b i l i t y of these substances to a l k y l a t e D N A I2, r a t h e r t h a n their a b i l i t y to produce either D N A p o l y m e r a s e I repairable s t r a n d breaks or c h r o m o s o m a l " c a t a s t r o p h e " . Since b o t h agents cause mutagenesis b y misrepairL it would seem reasonable to hope t h a t the lesions subject to misrepair might show up under a p p r o p r i a t e conditions as physicochem.ical d a m a g e to D N A , either as direct s t r a n d b r e a k s (cf. 7-irradiationS, ~") or as discontinuities in n e w l y - s y n t h e s i z e d D N A (@ UV irradiationI~). In fact MMS produces b o t h classes of damage. P r o v i d e d both are subject to equally inaccurate repair the " s t r a n d b r e a k s " in p a r e n t a l D N A in a polA ~ strain will be of less i m p o r t a n c e in m u t a g e n e s i s t h a n discontinuities in newly-synthesized D N A , since such " s t r a n d b r e a k s " are fewer a n d s u b j e c t to less repair. Discontinuities in newly-synthesized D N A could also be responsible for dichlorw}s nmtagenesis, b u t we would b a r e l y be able to d e t e c t t h e m on our g r a d i e n t s at ~.~15 lower yield. M a m m a l i a n mutagencsis In Chinese h a m s t e r cells, our d a t a show t h a t in the comparison between MMS a n d dichlorvos, s u r v i v a l correlates with the e x t e n t to which the agents a l k v l a t e protein 12. D N A s t r a n d b r e a k a g e on the other hand, correlates more with the e x t e n t of a l k y l a t i o n of D N A . It is not clear w h e t h e r the breaks observed on the g r a d i e n t exist as real or p o t e n t i a l s t r a n d breaks in the cell. As far as m u t a t i o n is concerned, MMS has been d e m o n s t r a t e d to act as a m u t a g e n for these Chinese h a m s t e r cells". We are aware ot no published d a t a cn dichlorw)s. Using dichlorw~s, TURNBULI~ (pers. comm.) has been unable to induce m u t a t i o n to 8-azaguanine resistance in our Chinese h a m s t e r cells u n d e r conditions where MMS was mutagenic. If, however, mutagenesis in m a m m a l i a n cells were to be correlated with the overall a l k y l a t i o n of D N A , dichlorvos might be e x p e c t e d to be a v e r y weak m u t a g e n , b a r e l y d e t e c t a b l e b y c u r r e n t tests. ACKNOWLEDGEMENTS
We would like to t h a n k Dr. P. D. LAWLEY (Pollard's W o o d Research Station, Bucks.) Mr. B. J. DEAN (Tunstall L a b o r a t o r y , Shell Research, Sittingbourne, Kent) a n d Dr. B. A. BRIDGES for v a l u a b l e advice a n d discussion. Mrs. S. STEVENS a n d Miss W. M U R I L gave e x p e r t technical assistance and Dr. W. J. H. GRAY c o l l a b o r a t e d in p r e l i m i n a r y e x p e r i m e n t s . W e t h a n k Dr. M. G. ORMEROD who devised the calculation given in the APPENDIX, and Dr. P. D. LAWLEY for permission to quote results before publication. REFERENCES I ARLETT, C. F.,
Mutation testing with cultured mamnlalian cells, Lab. Practice, 2J (1972) 420
424 •
M., AND N. SYMONDS,Radiation sensitive mutants of T4D. I.T4y: a new radiation sensitive mutant; effect of the mutation on radiation survival, growth and recombination, iViutation Res., 8 (1969) 431 439. 3 BRIDGES, B. A., AND R. P. MOTTERSHEAD, Gamma ray mutagenesis in a straiu of Escherichia coli deficient in DNA polynlerase I, Heredity, 29 (1972) 2o3-211. 4 BRIDGES, B. A., R. P. MOTTERSHEAD, M. H. L. (;REEN AND W. J. H. GRAY, M u t a g e n i c i t y of 2 BOYLE, J.
d i c h l o r v o s a n d m e t h y l m e t h a n e s u l p h o n a t e for Escherichia coli "WP2 a n d some d e r i v a t i v e s deficient in D N A repair, 3 l u t a t i o n Res., 19 (1973) 295 303 • 5 BUHL, S. N., AND J. D. REGAN, D N A r e p l i c a t i o n in h u m a n cells t r e a t e d w i t h n l e t h v l n l e t h a n c s u l p h o n a t e , M u t a t i o n Res., 18 (1973) 191-197.
DICHLORVOS AND MMS INDUCED DNA STRAND BREAKS
377
6 CHU, E. H. Y., AND H. V. MALLING, M a m m a l i a n cell genetics, II. Chemical i n d u c t i o n of specific locus m u t a t i o n s in Chinese h a m s t e r cells, Proc. Natl. Acid. Sci. (U.S.), 61 (1968) 13o6-1312. 7 F o x , B. W., AND M. FOX, S e n s i t i v i t y of t h e n e w l y - s y n t h e s i z e d a n d t e m p l a t e D N A of l y m p h o m a cells to d a m a g e b y MMS a n d t h e n a t u r e of associated " r e p a i r " processes Mutation Res. 8 (1969) 629-638. 8 GREEN, M. H. L., W. J. H. GRAY, S. G. SEDGWICK AND n . A. BRIDGES, R e p a i r of D N A d a m a g e p r o d u c e d b y g a m m a - r a d i a t i o n in Escherichia coli K- 12 a n d a r a d i a t i o n sensitive exrA d e r i v a t i v e d u r i n g inhibition of p r o t e i n s y n t h e s i s a n d n o r m a l D N A replication b y chloramphenicol, J. Sen. Microbiol., 77 (1973) 99-1o8. 9 KAPP, D. S., AND K. C. SMITH, R e p a i r of r a d i a t i o n - i n d u c e d d a m a g e in Escherichia cull, II Effect of rec a n d uvr m u t a t i o n s on radiosensitivity, a n d repair of X - r a y i n d u c e d single s t r a n d breaks in deoxyribonucleic acid, J. Bacteriol., lO 3 (197 o) 49-64. IO LAWLEY, P. D., AND P. BROOKES, C y t o t o x i c i t y of a l k y l a t i n g a g e n t s t o w a r d s sensitive a n d res i s t a n t s t r a i n s of E. cull in relation to e x t e n t a n d m o d e of a l k y l a t i o n of cellular m a c r o m o l e c u l e s a n d repair of a l k y l a t i o n lesions in D N A , Biochem. J., lO9 (1968) 433-447I I LAWLEY, P. D., AND D. J. ORR, Specific excision of m e t h y l a t i o n p r o d u c t s f r o m D N A of E. coli t r e a t e d w i t h N-methyl-N'-nitro-N-nitrosoguanidine, Chem.-Biol. Interactions, 2 ( 197 o) 154-157. 12 LAWLEY, P. O., S. A. SHAH AND D. J. ORR, M e t h y l a t i o n of nucleic acids b y 2-2 dichlorovinyl d i m e t h y l p h o s p h a t e , Chem.-Biol. Interactions, 8 (1974) 171-182. 13 LEHMANN, A. R., P o s t r e p l i c a t i o n repair of D N A in ultraviolet~irradiated m a m m a l i a n cells, J. Mol. Biol., 66 (1972) 319-337 . 14 LEHMANN, A. R., AND M. G. ORMEROD, An a r t e f a c t in t h e m e a s u r e m e n t of t h e molecular weight of pulse labelled D N A , Nature, 221 (1969) lO53-1o56. 15 LOFROTH, G., C. KIM AND S. HUSSAIN, A l k y l a t i n g p r o p e r t y of 2-2 Dichlorovinyl D i m e t h y l P h o s p h a t e : a d i s r e g a r d e d h a z a r d , E M S Newsletter, 2 (1969) 21-27. 16 MCGRATH, R. A., AND R. W. WILLIAMS, R e c o n s t r u c t i o n in vivo of irradiated Escherichia cull deoxyribonucleic acid; t h e rejoining of b r o k e n pieces, Nature, 212 (1966) 534-535. 17 OKAZAKI, R., M. ARISAWA AND A. SUGINO, Slow j o i n i n g of n e w l y replicated D N A c h a i n s in D N A p o l y m e r a s e 1-deficient Escherichia cull m u t a n t s , Proc. Natl. Acad. Sci. (U.S.), 68 (1971) 2954-2957. 18 PRAKASH, L., AND B. STRAUSS, R e p a i r of a l k y l a t i o n d a m a g e : Stability of m e t h y l g r o u p s in Bacillus subtilis t r e a t e d w i t h m e t h y l m e t h a n e s u l p h o n a t e , J. Bacteriol., lO2 (197 o) 76o-766. 19 RuPP, W. D., AND P. HOWARD-FLANDERS, D i s c o n t i n u i t i e s in t h e D N A s y n t h e s i z e d in an excision defective s t r a i n of Escherichia cull following u l t r a v i o l e t irradiation, J. Mol. Biol., 31 (1968) 291-3o4 . 20 SEDGWlCK, S. G., AND B. A. BRIDGES, Effect of p h o t o r e a c t i v a t i o n on t h e filling of g a p s in D N A s y n t h e s i z e d after e x p o s u r e of Escherichia coli to u l t r a v i o l e t light, J. Bacteriol., 117 (1974) lO77-1o81. 21 SETLOW, R. B., J. D. REGAN, J. GERMAN AND W. L. CARRIER, E v i d e n c e t h a t Xeroderma pigmentosum cells do n o t p e r f o r m t h e first step in t h e repair of u l t r a v i o l e t d a m a g e to their D N A , Proc. Natl. Acid. Sci. (U.S.), 64 (1969) lO35-1o41. 22 SINCLAIR, W. K., AND R. A. MORTON, X - r a y a n d u l t r a v i o l e t s e n s i t i v i t y of s y n c h r o n i s e d Chinese h a m s t e r cells a t v a r i o u s stages of t h e cell cycle, Biophys. J . , 5 (1965) 1-25. 23 STRAUSS, B. S., AND R. WAHL, T h e presence of baeaks in t h e D e o x y r i b o n u c l e i c acid of Bacillus subtilis t r e a t e d in vivo w i t h t h e a l k y l a t i n g a g e n t m e t h y l m e t h a n e s u l p h o n a t e , Biochim. Biophys. Acta, 80 (1964) 116-126. 24 STUDIER, F. W., S e d i m e n t a t i o n s t u d i e s of t h e size a n d s h a p e of D N A , J. Mol. Biol., i i (1965)373. 25 TOWN, C. D., K. C. SMITH AND S . S. KAPLAN, D N A p o l y m e r a s e required for t h e rapid rejoining of X - r a y i n d u c e d D N A s t r a n d b r e a k s in vivo, Science, 172 (1971) 851-853. 26 WALKER, I. G., AND D. F. EWART, T h e n a t u r e of single s t r a n d b r e a k s following t r e a t m e n t of L-cells w i t h m e t h y l a t i n g agents, Mutation Res., ~9 (1973) 331-341. 27 BEDFORD, C. T., AND J. ROBINSON, T h e a l k y l a t i n g properties of o r g a n o p h o s p h a t e s Xenobiotica, 2 (1972) 3o7-337 • APPENDIX
C a l c u l a t i o n o f n u m b e r - a v e r a g e molecular weights The number-average
Mn =
m o l e c u l a r w e i g h t of a p o p u l a t i o n
nMdM o
ndM •
o
of m o l e c u l e s is d e f i n e d a s
(z)
378
M.H.L. GREEN et al.
where n is the n u m b e r of molecules of molecular weight between M and M + d M . For a r a n d o m distribution of molecules (produced by the r a n d o m introduction of more than about five breaks per molecule into an initial population of molecules) n ~ A exp(--M/M~) where A is a constant (adapted from CHARLESBy1). Let us consider the apparent number-average molecular weight M'n obtained by integrating equation (z) from a finite lower limit M1, for a r a n d o m distribution. M,' =
M/Mn exp(-1
Mn)d
ex
--M/Mn)dM
M1
: M~ + M 1
(2)
i.e., to obtain the true value Mn, a correction factor equal to M1 must be subtracted from M'n. Applying the above to a discrete radioactivity distribution from the fractions in a sucrose gradient, equation (5) gives oo
oo
Mn = Z C , / Z C~/M,, i=o
(3)
i=o
where Ct is the radioactivity in a fraction of average molecular weight M, (assuming t h a t the molecules are uniformly labelled). Direct calculation of Mn from a radioactivity distribution from a sucrose gradient b y this summation is extremely inaccurate. The summation is strongly influenced b y fractions at the top of the gradient ; these are often contaminated with a great deal of spurious material (e.g. see Fig. 2). This causes the summation to be grossly in error. For a r a n d o m distribution of molecules, Mn can be accurately determined by a graphical method, which also tests the randomness of the distribution ~,~. Alternatively Mn m a y be calculated b y carrying out the above summation from a finite lower limit and applying the correction factor from equation (2). The graphical method demonstrated t h a t the radioactivity profiles obtained in our experiments were not in fact r a n d o m and it could not therefore be used to calculate Mn. Equation (2) is also only strictly valid for a r a n d o m distribution of molecules. However, the profiles we , were considering did not deviate very much from randomness below the top 4-6 fractions so t h a t errors in using the correction factor M1 should only produce a second-order error in the value of M,. This method has therefore been used for calculation of Mn although it is appreciated t h a t the calculations are not rigorously valid. REFERENCES (Appendix) A., Molecular weight changes in the degradation of long-chain polymers, Proc. Roy. Soc. (London), Ser. A, 224 (1954) 12o. 2 DEAN, C. J., M. G. ORMEROD,R. W. SERRIANIAND P. ALEXANDER,DNA strand breakage in cells irradiated with X-rays, Nature, 222 (1969) lO42. 3 LEHMANN, A. R., AND M. G. ORMEROD, The replication of DNA in murine lymphoma cells (L 5178Y) I rate of replication, Biochim. Biophys. Acta, 204 (197o) 128-143. I CHARLESBY,