chem.-Biol* zn~~fffcfions, 11 (1975) 193-17s @ Ebevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

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EFFECT OF PROCAINE HYDROCtlLBRIDE ON DNA REPAIR IN ~~C~E~C~IA CULI

C. K. K. NAIR AND D. S. PRADHAN

Biochemistry aad Food Technology Z&Vision, Ehabha Alomie Research Centre, Tromboy, Bombay-85 (India)

(Rcx&ed December 2nd, 1974) (Revision received February 1 lth, 1975) {Accepted February 20th, 1975)

SUMMARY

Liquid-holding recovery and r.:joining of y-radiation-induced DNA single- strand scissions in Escherichia coli could be effectively inhibited by procaine hydro- chloride at the concentration of 20 mM . At this cancentration, the drug also reversibly altered cellular permeability barrier 8s evidenced from the uptake of acriflavin by bacteria1 cells.

INTRODUCTION

The local anaesthetic procaine hydrochloride has been shown recently to enhance radiation-induced Iethality in bacterial cells, when added immediately after irradiationr. The radiosensitising aciion could be ascribed to inhibition of DNA repair by this chemical. In this paper, evidence has been presented to show that at the concentration of 20 mM, this drug arrests liquid-hording recovery and also rejoining of radiation-induced single-strand breaks in E.coZi B/r. Cell permeability was also found to be affected reversibly at this concentration of the drug an assessed by cellular uptake of acriflavin.

MATERIALS AND MFTHODS

Bffcterid strains Escherichiu coli B/r h&thy-tr !f- was obtained from Dr. D. Billen, University

of Florida (U.S.A.) and Escherichic r coli AB2487F- recA-thr-leu-arg-his-thi-pro- thy-ara-lac-gal-mtl-xyl-strr T6r 3 from Dr. K. C, Smith, University of Stanford (U.S.A.).

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Medium M9 mediums supplemented with 0.4% glucose, 4.0 Erg/ml thymine and 0.5%

casamino acids was used as growth medium for Ecoli B/r hcr+thy-try- cells. Thiamine hydr~hloride was added to this medium to the final concentration of 0.5 pg/ml for culturing Ecoli AB2$87 cells. Plating of bacteria was done on Difco nutrient agar, supplemented with 0.5 y0 yeast extract.

Chemicals Procaine hydrochloride was purchased from Hoechst Pharmaceuticals, Bom-

bay (India) and acriflavin was from Ffuka Adms, Buchs SG (Switzerland). [%&Me]- ~y~dine of specific activity 6 Ci~mmole, was obtained from Isotope Division, B.A.R.C., Bombay (India).

DNA sedimentation analysis Repair of single-strand breaks in DNA induced by y-radiation was studied by

sedimenting prelabelled cellular DNA in alkaline sucrose density gradients according to the procedure of McGrath and WilIiams3 as modified by Kaplan*.

liquid-Harding recovery Liquid-holding recovery of ultraviolet-irradiated Ecoli A82487 cells was in-

vestigated as described by Ganesan and Smiths.

Studies on acrijavin uptake The procedure of Silver and Wend@ was followed to measure the uptake of

acrigavin to determine changes in permeability induced in bacterial cells.

TIME OF INCUBATION, h

Fig. 1. Liquid-holding recovery in E.wli AB2487. Bacterial cells from exponentially growing cultures were exposed to 100 erg/mm2 ultraviolet light in M9 medium and incubated at 37” in the dark in growth medium lacking amino acids for various times. Viability was determined by plating in nutrient agar supplemented wrth 0.5 % yeast extract. a--@, control; A-A, 20 n-&f procaine hydrochloride.

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FRACTION NUMBER Bolbm

Fig. 2. Sedimentation profiles of DNA from y-irradiated (20 klad) E.co/i B/r her+ thy- try- cells after incubation in growth medium with or without 20 mM procaine hydrochloride. The bacterial DNA was prelabelled with [aH]thymidine by growing t’r . c dls b 17 h m growth medium containing 200 &i/ml [3H-Melthymidine (specific activity 6 Ci/mmole). The cells were exposed to 20 krad y-radiation under anoxia in 0.022 M phosphate buffer pH 7.0 and incubated in growth medium at 37” in the presence and absence of 20 mMprocaine hydrochloride. The cells were converted to sphero- plasts by the Tris-EDTA-lysozyme procedure314 and lysed with dupanol on 4.8 ml 520% linear sucrose gradient in 0.1 M NaOH and 0.9 M NaCI. The gradients were centrifuged at 30 000 rev./min for 90 min at 20” using SW65 rotor m a Spmco LZ -65 B ultracentrifuge and fractionated by syphoning. Trichloroacetic acid-insoluble radioactivity in each fraction was de&rminedA and expressed as percent of the total act&y loaded on the gradient. First moments (D:) and the sedimentation co- efiicients were calculated according to Kaplan4 and McGrath and Wlihams3.

RESULTS

E#ect of procaine hydrochloride on liquid-holding recovery Fig. 1 illustrates the effect of 20 mM procaine hydrochloride on liquid-holding

recovery in a uvr+ ret- mutant of E.coli. It is apparent that when E.coli AB2487 cells were exposed to 100 erg/mm2 ultraviolet radiation and incubated in the growth medium lacking amino acids at 37” in the dark, the survival of the culture increases with time due to repair of ultraviolet radiation-induced lesions in DNA5. This liquid- holding recovery is inhibited by procaine hydrochloride at 20 mM concentration.

Inhibition of repair of DNA single-strand scissions by procake hydrochloride Sedimentation profiles of DNA from E.coli cells exposed to 20 krad y-radiation

and post-irradiation incubation in the presence and absence of 20 mM procaine hydrochloride are illustrated in Fig. 2. The sedimentation coefficient of alkali- denatured single-stranded DNA of the bacterial cells decreased from IO!% to 87s after exposure to 20 krad y-radiation. Upon incubation in growth medium for 60 min at 37”, the sedimentation coefficient of DNA from irradiated cells increased to 98s

I I I I

10 20 30 bU PROCAINE HCI , mM

Fig. 3. Uptake of acriflavin in hwli ceils induced by different concentrations of procaine hydro- chloride. E.coli B/r hcit thy- try- 1~4s from exponentiauy growing cultures were incubate with different concentrations of procaine hydrochloride and 0.75 pg/ml acrifiavin in growth medium for 30 min at 37” at the dens&y of 108 cells per ml. The cell suspensions were centrifuged and acriflavin concentration in the supernatant was determined by measuring the fluorescence of the dye using a Hitachi Fluorescence Spectrophotometer Model 203. The fluorescence of 0.75 pg/ml acriflavin in growth medium was taken as 100%.

indicating restitution of most of the single-strand breaks. When procaine hydro- chloride at 20 mM ~on~ntration was included in the growth m~ium no such in- crease in sedimentation coefficient of DNA from irradiated cells could be seen upon incubation for 60 min. Prolonging the incubation period for an additional 60 min did not improve the DNA sedimentation coefficient either.

Changes in permeability to acrijfavin induced by procaine hydrochloride Uptake of acriflavin by Exoli c&s has been considered as an index of damage

to the cell membrane permeability barriers. The effect of various concentrations of procaine hydrochloride on cell membrane permeability is presented in Fig. 3. Un- treated control cells take up 4 % of acriflavin which might be due to the binding of the dye to the cell surface. Upto 1 mMconcentration of procaine hydrochloride no change in acriflavin uptake by the cells is observable. At 5 mM concentration of the drug, the acritlavin uptake is increased to the extent of 12%. On further increasing the concen- tration of the drug, there was a graded enhan~ment in acriflavin uptake which reached 30% at 30 mM ~on~ntration of procaine hydr~h~oride,

Reversibility of procaine hydrochloride-induced permeability to acriflavin In Fig. 4, results of the uptake of acriflavin by the cells pre-incubated with

procaine hydrochloride (20 mM) for 60 min are shown. It can be seen that on incuba- tion of bacti;riaf c&s (pre-treated with procaine hydrochloride) in growth medium

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%k7%-&--- 0 50

TIME OF INCUBATION IN ABSENCE OF PROCAINE HCI, Mm

Fig, 4. Reversal of procaine hydrochloride induced acriflavin uptake by E.coli. E.coIi B/r her+ thy- try- cells from exponentially growing cultures were incubated with 20 mM procaine hydrochloride at 37” in growth medium for 30 min at the density of 10s cells/ml. The cells were harvested by milli- pore filtration from the treated cell suspensions and Incubated in growth medium devoid of procaine hydrochloride. At variaus times acrlflavin was added to a final concentration of 0.75 pg/ml and incubated at 37” for 30 min and centrifuged. The concentration of acriflavin in the supernatant was determmed as in Fig. 3.

devoid of the drug at 37” for 10 min, the acriflavin uptake is restored to the control level.

DISCUSSION

Liquid-holding recovery of ultraviolet-irradiated bacteria has been postulated

to take place by the same mechanism as excision repair which invohes: (Q excision and degradation of small portions of the damaged DNA strand, (ii) repair replication in the excised region to fill in the gap; and (iii) rejoining of the newly synthesised portion to the main DNA strandv. It has been suggested that repair of DNA damage induced by ionising radiations could also take place by the same even&r-g. The inhibition of liquid-holding recovery and rejoining of y-radiation-induced single-

strand scissions by procaine hydrochloride could arise from blocking of a reaction common to both the processes presumably the steps (ii) and (iii) of the excision repair mentioned above.

The results presented in Figs. 3 and 4 reveal that procaine hydrochloride at the concentration of 20 mM has also a reversible effect on the cellular permeability barrier and is therefore indicative of alteration in the bacterial ceil membrane struc- ture. Studies with a synthetic model membrane suggest that procaine hydrochlcride may cause alterations in structural characteristics of membranePJ1. There have been a number of reports in recent years to show that in bacteria, DNA is attached at many points to the cell membrane 13~13. If the attachment of DNA to the cell mem- brane or the structural integrity of the cell membrane is essential for the repair of DNA damage, this process could be inhibited by agents acting on the cell membrane,

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such as procaine hydrochloride. We have recently found that phenethyl alcohol, a chemical known to affect the cell permeability barriers, also inhibits rejoining of y-radiation-induced single-strand scissionsr4.

Since procaine hydrochloride breaks down the cellular permeability barrier, it is possible that the block in DNA repair may arise from leaking out of small molecules or metal ions essential for this process.

Procaine hydrochloride may also be thought of as exerting its effect on DNA repair indirectly by overall suppression of cellular metabolism. We consider such a possibility unlikely since it is observed that procaine hydrochloride at the concentra- tion that completely inhibits repair of DNA allows syntheses of macromolecules though at somewhat reduced rates.

REFERENCES

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2 K. G. Lark, T. Repko and E. J. Hoffman, The effect of aminoacid deprivation on subsequent deoxyribonucleic acid replication, Biochim. Biophys. Acm, 76 (1963) 9-24.

3 R. A. McGrath and R. W. Williams, Reconstruction in viva of irradiited Ekherichia coli de- oxyribonucleic acid: rejoming of broken pieces, Nurrr, 212 (1966)534-53X

4 H. S. Kaplan, DNA strand scission and loss of viability after X-irradiation of normal and sensitized bacterial cells, Proc. Nurl. Acud. Sci. (US.), 55 (1966) 1442-1446.

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13 B. H. Rosenberg and L. F. Cavalieri, Shear sensitivity of the E.cofi genome: Multiple membrane attachment poirdts of the Exoli DNA, Cold Spring Harbor Symp, Quanr. B/of., 33 (1968) 65-72.

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