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Mutation Research, 33 (1975) 107--112 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

THE LETHAL EFFECT ON BACTERIA OF DIMETHYLNITROSAMINE USED WITHOUT AN ACTIVATING AGENT

JOHN READ

Radiation Biology Research, Cancer Society of New Zealand, Wakari Public Hospital, Dunedin (New Zealand)

(Received February 26th, 1975) (Revision received June 25th, 1975) (Accepted July 25th, 1975)

Summary

Survival curves have been obtained for various strains of bacteria treated with dimethylnitrosamine (DMN) solutions of different concentrations. The results are compatible with the conclusion that DMN kills the bacteria by an attack on their deoxyribonucleic acid (DNA) and the damage so caused can be repaired by the same systems that repair damage created by ultraviolet light or X radiation. No activating agent was added to the DMN solutions and under these circumstances their activities were in proportion to the squares of their concentrations. It is suggested that this is because two molecules of DMN plus one of oxygen produce two carbonium ions, two hydroxyl ions, two formalde- hyde molecules and two nitrogen molecules.

Int roduct ion

Today there is impressive evidence that oncogenic agents act by attacking the cells' DNA. The repair systems then function to put this right but in some cases the repair is faulty and malignancy may result. Dimethylnitrosamine is a powerful oncogenic agent. The original purpose of this work was to test its lethal action on various types of bacteria with very different capabilities for the repair of damage to their DNA. The survival curves so obtained could then be compared with the known survival curves following treatments with UV or X radiation. DMN does not produce cancer directly but only after it has been broken down by cell enzymes. The active agent is almost certainly the car- bonium ion. When DMN is used in an in vitro system it is usual to add an activating mixture. For example Laishes and Stich [3] used microsomes from mouse liver, NADPH, magnesium chloride and glucose-6-phosphate. Nothing of

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this kind was used in my experiments. If any enzyme system activated the DMN it must have been in the bacteria at the time of t reatment . In order to kill an appreciable propor t ion, concentrat ions of DMN of up to 1 molar were neces- sary which perhaps indicates that the bacteria were relatively inactive.

Materials and Methods

The bacteria used were Escherichia coli B/r, E. coli Bs_l, E. coli strain 62 and strain 64, and Salmonella typhimurium strains 35 and 37*. The strain num- bers have been used in this laboratory and will be retained in the body of this paper for brevity.

Strains 62 and 64 are identical except that strain 64 has a k prophage in- tegrated in the genome. Strains 35 and 37 are identical but 37 contains a Col I factor which increases its resistance against UV and X radiation compared with that of 35.

Bacterial culture In all cases the bacteria were cultured overnight at 37°C in Difco brain--heart

infusion. Next day two tubes each containing 5 ml of the same medium were inoculated with 0.05 ml of this culture. The two tubes were placed in a water bath at 37°C for 2 h which produced a cell density of about 2 × 108 per ml. The cells were spun down and washed three times in Dulbecco A phosphate- buffered saline, pH 7.3, and finally suspended in 10 ml of the same buffer. As oxygen is essential for the action of DMN this buffer had already been saturated with it.

Trea tm en t The suspension was divided into two equal parts and sufficient DMN {Aldrich

Chemical Company) was added to one to make the chosen molarity. An equal volume of buffer was added to the other part. The two samples were immersed in a water bath held at 37°C in a fume chamber for 1 h. The action of the DMN was stopped by extensive dilution with buffer. Finally the fraction which sur- vived the t rea tment was determined from the colony counts obtained by the usual pour-plate method followed by overnight incubation.

Results and Discussion

Both Lijinsky et al. [7] and Lawley et al. [6] have found a number of dif- ferent methyla ted bases in the DNA of rat liver cells isolated 5 h after adminis- t rat ion of dimethylni t rosamine to rats, so that it is known that DMN acts as an alkylating agent. Fur thermore Lijinsky et al. [7] used DMN whose hydrogen had been replaced by deuter ium to a greater extent than 97% and it was proved that the intact CD3 group was transferred to the DNA base. It has been sug-

* Stra in 62 is Escherichia coli K 12 ( H o w a r t h ) [1] ; s t r a i n 6 4 is Escherichia coli K 12 (k) ( H o w k r t h ) [1] ; s t r a i n 3 5 i s Sa lmone l la t y p h i m u r i u m L T 2 trp D1 ( H o w a r t h ) [2] ; s t ra in 37 is Salmone l la t y p h i m u r i u m L T 2 trp D1 IB-P9 ( H o w a r t h ) [2 ] . T h e s e s t r a i n s w e r e a gift f r o m Dr. Sheila T h o m p s o n of the Microbiol- ogy D e p a r t m e n t , Otago U n i v e r s i t y . f o r w h i c h I g i v e thanks .

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gested that DMN acts by forming diazomethane but this observation disproves that. We may safely assume that the DNA of the bacterial strains used in this experiment also suffered alkylation of their bases. Lawley and Brookes [4] treated E. coli strain B/r and strain Bs-1 with mustard gas, di-(2-chloroethyl) sulphide radioactively labelled with S 3 s. They showed that the alkylated DNA from strain B/r lost its radioactivity when the cells were incubated in growth medium. This action could be stopped by the addition of iodoacetamide which led them to the conclusion that the removal of the radioactivity was enzyma- tically mediated. On the other hand no radioactivity was lost from the bacteria of strain Bs-1 which showed that these cells were unable to excise the alkyl groups.

A similar experiment was described by Lawley and Brookes [5]. Removal of alkyl groups from DNA of the cells of resistant strains B/r and 151- was demon- strated but the sensitive strain Bs-z only did so at very low extents of alkyla- tion. Lawley and Brookes also gave survival curves for cells of the various strains after treatments with mustard gas, half sulphur mustard, or methyl methanesulphonate. The resistant strains all had curves with shoulders, i.e. the initial slope at low doses was much less than the slope at large doses because these strains had efficient mechanisms for excising alkyl groups and rebuilding the DNA provided the treatments were not too severe. However the survival curve obtained with Bs_ 1 was a straight line through the origin in every case.

The survival curves for the E. coli strains after the DMN treatments are shown in Fig. l a and for the Salmonella strains in Fig. lb . Fig. l b should be

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Fig. l a . T h e survival of 4 s t rains of E. col i t r e a t e d for 1 h at 37°C, p H 7.3, in o x y g e n s a t u r a t e d DMN solu- t ions. The abscissa scale h a s b e e n g r a d u a t e d in p r o p o r t i o n to the square of the c o n c e n t r a t i o n of DMN b u t t h e n u m b e r s e n t e r e d are t h e a c t u a l c o n c e n t r a t i o n s .

Fig. l b . T he survival of 2 s t rains of S. t y p h i r n u r i u m t r e a t e d f o r 1 h a t 37°C, pH 7.3, in o x y g e n - s a t u r a t e d DMN solut ions . T he abscissa scale h a s b e e n g r a d u a t e d in p r o p o r t i o n to the square of t h e c o n c e n t r a t i o n of DMN b u t t h e n u m b e r s e n t e r e d a r e t h e a c t u a l c o n c e n t r a t i o n s .

110

10 ( .colt o - f l 8 2 5 )

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U V d o s e ( ra in ) Fig. 2. T he survival of the m u t a n t tryD-1 of S. typhimurium L T 2 and of col ic inogenic der iva t ives car ry- ing Col I fac tors f r o m d i f f e r en t sources a f te r t r e a t m e n t s w i th doses of u l t rav io le t rad ia t ion . This figure has been r e p r o d u c e d f r o m a pa pe r by H o w a r t h - T h o m p s o n , Proceed ings of the Otago Medical School , 44 No. 1, Apri l 1966 , 5.

compared with Fig. 2 which is reproduced from a paper by Howar th-Thompson (see legend). The Col I factor has reduced the sensitivity to DMN by a factor of 1.25. Read [9] treated the same two Salmonella strains with X rays and the corresponding difference in sensitivity was 1.27. However Fig. 2 shows that when UV light was used the factor was about 3.

The curves obtained with the E. coli strains are similar to those obtained by Lawley and Brookes. The resistant strains produce curves with shoulders where- as the curve for Bs-1 is a straight line through the origin. That is just as this strain is unable to excise thymine dimers produced by UV and alkylations by mustard gas and half sulphur mustard so it is unable to excise some additions to the DNA made by the DMN, almost certainly alkyl groups. The impor tant result of this work is that this correspondence between the shapes of the sur- vival curves and those shapes which a wide experience has led us to expect is only obtained if the log survivals are plot ted against the squares of the con- centrat ions of DMN. If, for example, the results for strain B~-I are plot ted against the first power of the concentrat ions a curve with a shoulder is obtained which lies very close to the curve for B/r as plot ted on Fig. la . It is suggested that in the case of this exper iment where no activating mixture was used the extent of the action depended on the square of the concentra t ion of DMN be- cause two DMN molecules interacted so:

CH3~ 2 CH3~'N--NO + 02 -~ 2CH~ + 2OH- + 2HCOH + 2N2

This would provide the carbonium ions to effect the alkylation. This is sup

1 1 1

ported by the results of an experiment done by Dr. R.J. Wilkins in this labora- tory. He suspended log phase E. coli B/r bacteria at a concentration of l 0 s per ml in oxygenated minimal salts solution made one molar in DMN. They were incubated for 1 h at 37°C, pH 7.0, so that the t reatment was virtually the same as those used in the lethality experiments. The cells were then washed and lysed on an alkaline sucrose gradient and centrifuged. Single strand breaks in the denatured DNA were detected by the method of McGrath and Williams [8]. The average molecular weight of the DNA fragments was 1/50 of that of bacteria similarly treated but with the DMN omitted. When the bacteria were treated with a 1/10 molar DMN solution the DNA showed no detectable reduc- t ion in molecular weight. If the number of breaks produced had been in direct proportion to the concentrations a change could have been detected. This suggests that the breaks were produced in proportion to a power of the con- centration greater than uni ty in line with the conclusion that the lethal effect depends on the square of the concentrations.

The points for strains 62 and 64 lie about the same survival curve so the possession of the k prophage by strain 64 had no influence on the sensitivity to DMN. Dr. Sheila Thompson of the Microbiology Department, Otago University, was unable to detect any induction of the lambda phage by the standard DMN treatment.

References

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