Initial Degradation of Deoxyribonucleic Acid after ...(6, 13, 16, 21, 29), this parameter has been measured directly by viscosimetry after i.v. or i.p. injection of DNA. Parallel chemical

Study of the fate of DNA immediately after its administration in mammals is important since specific biologiceffects may be induced very early, namely, transformation,infectivity, and neoplasia.

Generally, papers published in this field (8, 24, 27) arebased upon methods which do not always allow distinctionbetween the initial macromolecules injected and theirdegradation products. Since it is known that biologicactivity of DNA is related to its degree of polymerization(6, 13, 16, 21, 29), this parameter has been measureddirectly by viscosimetry after i.v. or i.p. injection of DNA.Parallel chemical determinations were also made.

In an attempt to correlate the DNA catabolism and theDNase plasma activity, these experiments were done onindividual rabbits, in which the blood enzymatic activityvaried greatly from one animal to another, and on differentinbred strains of mice, each strain having a constant and

1 This work has been done under Contract 61-FR-060 with the

ComitéCancer et Leucémiede la DélégationGénéralea la Recherche Scientifique et Technique.

2 The following abbreviations are used : DNA, native deoxy

ribonucleic acid as extracted by usual methods; insol-DNA, polydesoxyribonucleotides insoluble in perchloric acid (PCA) underusual conditions; sol-DNA, oligonucleotides soluble in PCA (wedid not find a report or publication of the M.W. of polydeoxyribonucleotides and acid precipitability, but, one can supposethat the M.W. is low since an exhaustive enzymatic attack of DNAis necessary before the appearance of sol-DNA is observed; DNase,neutral deoxyribonuclease(s) of blood plasma; MG, methylgreen; KU, Kurnick unit (10) for neutral DNase activity; [@l,intrinsic viscosity expressed in c.g.s. units; €P,molar coefficientof extinction (260mg) calculated from phosphorus concentration.

Received for publication October 2, 1964; revised February 3,1965.

genetically conditioned DNase activity different from theother and little variation among animals.3

Moreover, the DNA catabolism has been followed inthe presence of substances (Na-citrate, MG) which areknown to inhibit DNase activity (12). The results indicate that the period throughout which an exogenous DNAwould maintain in the blood a physical state compatiblewith any specific biologic potency is very short—not morethan a few mm ; they also show that the plasma DNasesplay a nearly exclusive part in the initial fate of DNA,only a very small fraction of native macromoleculesdiffusing out of the blood stream. Some of these experiments have been previously reported briefly (19).

MATERIAL

DNA.—DNA extracted from calf thymus according tothe method of Kay et al. (9) (Sample a) had M.W.,5.2 X 106; [@] 3200; eP, 6800; according to the method ofSigner and Schwander (23) (Sample b), M.W., 6.5 X 10@;[‘7],6200; €P,6900; and according to the method of Aubimet al. (1) (Sample c), M.W., 6 X 10@;@ 5200; €P,6600.

M.W. was measured by light scattering with an apparatus of the C.R.M. Strasbourg type according to Wipplerand Scheibling (28). DNA solutions were purified bycentrifugation for 5 hr at 30,000 X g (Servall, refrigerated).Phosphorus and protein contents were determined according to Dryer et a2. (4) and Lowry et al. (18), respectively ; all the samples contained <0.5 % protein. Viscosimetric determinations were done as described below.

$ Paoletti, C., Pascaud, X., Auger, P., Gosse, Ch., and Tru

haut, R., to be published.

877

Initial Degradation of Deoxyribonucleic Acid after Injection in

Mammals1

CH. GOSSE, J. B. LE PECQ, P. DEFRANCE, AND C. PAOLETTI

(Laboratoire de Biochimie et Enzymologie et Laboratoire d' Ultracentrifugalion, Institut Gustave Roussy, Villejuif (Seine), France)

SUMMARY

The fate of DNA2 in the blood of rabbits and mice, after i.v. and i.p. injection,has been followed by viscosimetry and chemical determination of acid-precipitableDNA. It has been shown that the initial degradation of DNA is due to neutralDNase activity, which sets up a very effective biochemical barrier against exogenousDNA. The change in molecular weight of DNA administered in vivo has been calculated and correlated with the time after which physical properties of DNA are notconsistent with biologic activity. This time can be expected to vary from a few secto about 10 min in animal species with low plasma DNase level, such as man. Itcan be increased by using DNase-inhibitors like methyl green.

Although it is unlikely that a large amount of highly polymerized DNA couldreach cells without previous depolymerization after i.v. administration, our resultsare compatible with the expression of any infecting or transforming properties ofthis material.

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878 Cancer Research Vol. 25, July 1965

Methyl Green (Merck).—Solutions in 0.14 M NaCl werepurified by several chloroform washings.

DNA-Methyl Green Complex (DNA-MG) Solution.—DNA (2 mg/mi), 45 ml; 0.2 % MG, 45 ml; Tris-HC1 buffer, 0.4 M, pH 7.4, 10 ml, were mixed.

METHODS

1. Chemical Determinations of Insol-DNA and Sol-DNAin Blood Plasma.—These were done according to a slightlymodified Schmidt-Thannhauser method (22). Plasmawas treated with twice its volume of PCA (9 %) at 4°C;after centrifugation, sol-DNA was found in the supernatant. Precipitates which contained insol-DNA werewashed with PCA (9 %), hydrolyzed twice for 30 miii in 1volume of PCA (0.5 M) at 70°C, and then washed withPCA (0.5 M). Analysis of deoxyribose was made in eachfraction by the Dische-Burton method (2), pure deoxyribose (National Biochemical Corporation) being used asa standard.

@. Treatment of Animals.—We used 3-kg rabbits of the

strain “Fauve de Bourgogne,― 6—8months old, whoseplasma DNase activity varied from undetectable to about0.20 KU, and 3 strains of mice with a wide range of DNaseactivity : strain C3H (0.12 ± 0.01 KU) ; strain Swiss(0.21 ± 0.02 KU), whose characteristics are describedelsewhere (3) ; and strain XLII (0.29 ± 0.02 KU), raisedby inbreeding by the Centre National de Selection d'Animaux de Laboratoire, Gif-sur-Yvette, and bred in ouranimal department for 22 generations.

a) DNA Administration i.v.—In 1 experiment, 4 rabbitshaving comparable plasma DNase activity (0.11 KU) werechosen ; they received 3, 5, 10, and 20 mg of DNA. Inanother experiment, 21 rabbits whose plasma DNaseactivity varied from 0.03 to 0.18 KU received 10 mg eachof DNA. Individual blood sampling was done at regularintervals by heart puncture. DNase activity did notvary from one sample to another.

Twenty-five to 30 mice were selected in each strain;each mouse received 200 @tgof DNA; groups of 5 animalswere sacrificed at chosen intervals ; pooled blood sampleswere gathered to determine DNase activity either inheparin or trisodium citrate (0.2 ml of a 0.33 M solution)or for viscosimetric or chemical determination.

To test the role of DNase inhibitors, 2 series of experiments were designed : in the 1st one, 3 rabbits received10 i.v. administrations of 30 smoles of trisodium citrateover 60 mm, 10 mg of DNA being injected immediatelyafter the 1st citrate injection ; in the 2nd one, 5 to 7 mlof a solution of 0.2 % MG were injected i.v. ; immediatelyafterwards, 11—13ml of DNA-MG complex were given,and finally a slow perfusion of MG (20 mg in 15 mm) wasmade to decrease the rate of complex dissociation bykeeping the free dye concentration as high as possible.

DNA solutions were injected in the marginal vein of therabbit ear and in the tail veins of mice. DNA did notelicit any symptom of toxicity. MG is markedly toxic.

b) DNA Administration i.p.—Swiss mice were used,each animal receiving 0.5 ml of a solution of DNA, 2mg/mi, or 1 ml of a solution of DNA-MG previouslydescribed. Immediately after injection and at regular

intervals blood samples were removed from the retroorbital sinus.

3) Determination of Plasma Neutral DNase Activity.—This was done according to the method of Kurnick (10);a plasma containing 1 Kurnick unit/mi has the sameenzymatic activity as a crystalline pancreatic DNaseWorthington solution of 5 @ig/ml.

4) ViscosimetrivDeterminations.—Determinationsweremade before and after DNA injection on 2 ml of citratedplasma in a Couette viscosimeter, Lecomte de Nouy type(17). Results are given as specific viscosity (@ sp) andreduced visosity@ red),4 normal plasma being used assolvent.

This apparatus allows very sensitive measurements ofviscosity at a low rate of shear (15 sec@). A variation oflisp from 0 to 0.05, roughly corresponding to 5—10 @g/mlof native DNA, is readily detected.

As a first approximation, [@@]is proportional to theM.W. of DNA when degraded by DNase. As shown byThomas (25)

En]= K'M (A)

On the other hand, @redis related to [@@]by the Hugginsequation

@red= [ui + k[@]2c (B)

where k is a constant for a given DNA.If k, M.W., and [nl of the DNA initially used are

measured, these 2 equations permit the calculation of theM.W. of the DNA in the animal blood plasma from themeasures of @redand c.

When DNA is degraded further—[@} < 2000—EquationA becomes: @red= [,@](11).

The initial slope K (decrease of @sp/min) of the curvelisp = f(t) determined in vivo allows, under these conditions, the estimation of the rate of depolymerization(VD) of the DNA (11).

Although the animals received the same quantity ofDNA, the initial ijsp varied slightly from one animal toanother. To compensate for this variation, VD is givenas K/@spo.

RESULTS

1. Fate of DNA after i.v. Admini.stration.—For eachrabbit, the decline of insol-DNA in plasma was a linearfunction of time, and consequently the quantity of insolDNA which disappeared per unit of time was constantand independent of the quantity of insol-DNA actuallypresent at each time. The rate of disappearance of insolDNA (V) expressed as @gof insol-DNA/min/ml appearedto be proportional to the quantity of DNA injected, i.e.,to the initial concentration of DNA (Chart 1). Althoughthese results are apparently contradictory, an indirectexplanation for them can be offered (see below under“Discussion―). Chart 2, A, allows a comparison betweenthe specific viscosity of plasma and the concentration ofinsol-DNA versus time; specific viscosity decreased about

4 ‘15P = (nc ‘io)/'io where ‘7c@5the viscosity of a solution of

concentration c and ,j@is the viscosity of the solvent ; ,@red =‘7sp/c and [,@ = urn ,, red.

c-@O

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GOSSE et al.—Initial Degradation of DNA 879

amounts of insol-DNA appeared in mouse blood (Chart7). The highest concentration of insol-DNA and solDNA occurred at the same time as the highest viscosity,i.e., about 1 hr after injection of DNA. At this time, thereduced viscosity of blood was 1100 c.g.s., correspondingto insol-DNA of about 1 X io6 M.W. Complex formationwith MG markedly slowed the passage of deoxyribonucleicmaterial from peritoneum to blood, and the highest concentration of insol-DNA was observed only after 3 hr.

DISCUSSION

The plasma DNases play a fundamental and probablyexclusive role in the initial degradation of DNA. Threeexperimental observations support this conclusion.

a) During the 3 mm after DNA injection in blood thereis a great deal of difference between the rapid decreaseof viscosity indicating a depolymerization of DNA andthe constant level of insol-DNA in plasma (Chart 2).Such data exclude the hypothesis of a rapid and importantescape of native DNA out of vascular spaces during thisinitial step and demonstrate that the only significantphenomenon is the breaking down of phosphodiesterlinkages of DNA; this can only be due to enzymaticattack since shear degradation in the capillaries is verylikely inoperative owing to the relatively low DNA M.W.(@6 X 1o@).

b) There is a proportionality between the initial rate ofdepolymerization (VD) and the plasma neutral DNaseactivity level (Chart 6), a proportionality which is alsofound with the disappearance of insol-DNA in mice (Chart5), as well as with the urinary excretion after i.v. injection of DNA-@-aminoisobutyric acid (Baiba), a specificcatabolite of DNA-thymine (26).

c) The disappearance of the DNA-MG complex isremarkably slow when compared with the disappearanceof DNA alone. It is unlikely that the DNA-MG complex,having a shape and a size similar to DNA, would diffuseout of vascular spaces at a diminished rate on mechanicalgrounds only ; the most satisfactory hypothesis to explainthis phenomenon is the strong DNase inhibition by MG.

Depolymerized, but stifi acid-precipitable DNA, produced from the initial depolymerization of native DNA isalso subject to DNase action, but in this case, such anenzymatic action is associated with a diffusion out ofvascular spaces. This is proved (a) in the rabbit by therate of disappearance of insol-DNA versus DNase activity(Chart 4) extrapolated to zero activity, where it is stillimportant (about 0.7 pg/mI/mm) ; (b) in mice by thepassage, in reverse, from peritoneum to blood of insolDNA.

The insol-DNA diffusion out of the blood space isslower in mice than in rabbits. This difference probablyexplains the absence of sol-DNA and the apparent linearityof the rate of insol-DNA disappearance in rabbit blood:diffusion of insol-DNA would be higher during its degradation and thus would balance the slowing down ofenzymatic degradation resulting from a decrease in substrate concentration.

Like plasma DNases, DNases from tissues seem to beactive in the catabolic fate of injected DNA since theappearance of insol-DNA in plasma after intraperitoneal

mad DNA@@“@/mI01 plasma)

1@

0 20 40 60time (mm)

CHART 1.—Disappearance of insol-DNA in rabbit plasma withidentical DNase activity (0.11 KU) after i.v. injection of variablequantities of DNA (3, 5, 10, and 20 mg) (Sample a).

50% in less than 3 ruin, whereas the insol-DNA concentration did not vary.

In mice, the rate of disappearance of insol-DNA wasconstant in the first minutes following i.v. injection ofDNA, but slower later (Chart 3).

This rate is correlated with the neutral DNase activityof plasma, both in rabbit and in mice (initial rate) (Charts4, 5). The correlation coefficients between the 2 variables,vi2. 0.88 and 0.98, are highly significant. The regressionline intersects the ordinate very near the origin in thecase of mice; in the case of rabbits, the regression linereached the ordinate far from the origin. Extrapolationgives an appreciable rate of insol-DNA disappearance forno DNase activity. But when the rate of DNA depolymerization (VD) is expressed versus DNase activity, incontrast to what is observed when the rate of insol-DNAdisappearance is plotted, a straight line going through theorigin is obtained (Chart 6). Sol-DNA was not found toappear before or after injection of DNA, or of endogenousinsol-DNA in rabbit plasma, for any DNase activity, atleast at the level of sensitivity of the method (5 j@g/ml).On the other hand, sol-DNA (15-20 @sg/m1)was found inmouse plasma 10-20 mm after injection of DNA foreach strain of mice used.

Injection of trisodium citrate did not significantlymodify the rate of insol-DNA disappearance. After injection of DNA-MG (Chart 2, B), the insol-DNA concentration did not change much during the first 10 min andthe plasma specific viscosity decreased very slightly.Such experiments must necessarily be short owing totoxicity of MG in doses needed to prevent dissociation ofthe DNA complex.

@d.Fate of DNA after i.p. adminz@tration.—Comsiderable

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ti@@

@@@@ A

A

,@

1.5

880 Cancer Research

lnsol. DNA(A1Wmiof plasma

200

0

I00

______Specificviscosity

—@ —- Insol. DNA

0 15 20time (mm)

15 20time @rnmn@

CHART 2. Comparative study of the specific viscosity and insol-DNA concentration versus time in rabbit plasma (DNase activity0.11 KU). (A) after i.v. injection of 10 mg of DNA (Sample c); (B) after i.v. injection of the complex DNA-MG (Sample c). i redare, respectively, in each case: (A) 7200,4600,3500,2200,1400,and 250; (B) 9500,10400,10100,and 8900(c.g.s.). In the case of freeDNA (A) : [‘iiwas 5000, 3500, 2800, 2200, 1400, and 250, respectively; and M.W. was 6 X 10', 4.7 X 10, 3 X 10', 2.6 X 10, 1.7 X 10', and2.7 X 10', respectively.

Insol. DNA

‘4.4

.4.4

.4

@@@4%@@@

% %

‘@@X(CH))) 3

80 time(min)40 60

CHART 3. Disappearance of insol-DNA in the plasma of 3 isogenic strains ofmice after i.v. injection of 200 @gof DNA (Sample c).

Vol. 25, July 1965

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xx

GOSSE et al.—Initial Degradation of DNA 881

injection of the relatively DNase-insensitive DNA-MGcomplex is delayed.

The existence of DNase inhibitors (5, 15) deserves somecomment. Such inhibitors are released from blood cellsin the plasma when the blood is withdrawn (7), but inmost cases they are too dilute to influence the enzymatic

Vc,uginsolDNA-‘mlof plasma@1@)

V(,@ mnsolDNA@mIOIplasma@@jfl)

kinetics in the 1\urnick in vitro experinwnlal conditions.Nevertheless, in a few cases, their (oncent ratioii could behigh enough, 111spite of the dilution, to slow the rate ofDNase action; this gives the most likely explanation ofthe 3 points found off the regression line iii Chart 6, sincethese points correspond to the 3 samples of lower DNaseactivity. Owing to the presence of such inhibitors, anextrapolation from an in vii'o experiment to an in viti@oone is difficult (27).

Supposing some analogy between the modalities ofaction of bacterial transforming DNA and viral or cellDNA's which would have specific biologic activity in anappropriate cellular system, one can estimate approximately the minimum M.W. necessary for a DNA to retainan eventual biologic potency when it diffuses in bloodcirculation and, consequently, evaluate the time duringwhich such an action can be expressed.

One hundred breaks on the molecule reduce the M.W.from 6 X 10@to 4 X 10@(25) and, 011 the other hand,reduce the biologic activity to 0.1@ of the initial value(6, 21). For a 0.10-KU I)Nase activity, about 3 mm areneeded to reach such a DNA M.W. in rabbit plasma afteri_u. injection of DNA.

If it is admitted that the biologic activity is in closeinverse relationship with the nuniber of phosphodiesterlinkages broken per molecule, from that observation thetime necessary to obtain any given decrease of such anactivity can easily be calculated. For example, 1 mmafter DNA injection, 90 % of any possible biologic activitywould be lost . This conclusion confirms the data ofHerriot (7), who found that a transforming DNA loses90% of its activity in a 12-miii contact with a DNase solution with an activity the san e as that of human plasma,which is about 10 times less active than rabbit plasma.

xx

xx

x

xx

x

1.5

1.0

0.5

0_ o.ôs o@o 015DNase activity (KU)

CHART 4. Rate of disappearance of insol-DNA in the plasma of21 rabbits after i_v. injection of 10 mg of DNA (Sample c) versustheir DNase activity. The initial concentration of DNA variedslightly in each animal ; rates refer to a single initial concentration.

6

5

4

3

2

I

++

‘4

,@ip#/ +CH

a.

0 0.10 020 0.30 DNaseACtiVitY (KU.)

CHART 5. Initial rate of disappearance of insol-DNA in plasma of 3 isogenicstrains of mice after i.v. injection of 200 @&gof DNA (Sample c) versu8 their DNaseactivity. The regression lines have been calculated.

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‘aI

‘a‘aS

882 Cancer Research

On the other hand, our data (Chart 1) show that the rate ofdegradation, expressed in proportion to the total numberof breaks occurring per ml and per miii, is roughly proportional to the initial DNA concentration ; this meansthat the iiumber of breaks per molecule is, for any DNAconcentration, constant for a given DNase activity, ascould be deduced from the kinetic studies on DNase (14).Those observations validated our conclusions for a largerange of DNA concentrations, at least up to about 500j.@g/mi; such concentrations obviously correspond to

0

CHART 6. Initial depolymerization rate of 1)NA, ITD , versusplasma l)Nase activity in 14 rahbits after iv. injection of 10 mgof l)NA (Sample b).

amounts of DNA well below those that can be injectedinto mammals.

Our results do not exclude the ability of DNA to expressits biologic potentialities in mammals after injectioli ; evena small percentage of the injected material containsenough DNA molecules of a 1\I.W. of 6 X 10@to transformor infect most of the animal's cells and a low but significantamount of high M.W. DNA which, because of the lack ofaccuracy of usual technics for measuring blood volume,might have been absorbed from the plasma before the 1stdetermination.

Finally, these results emphasize how crucial the choiceof species is when one tries to reveal biologic propertiesof DNA, since plasma 1)Nase activities vary in a largerange (5 X 10—sKU for man up to 2 KU for cat) (20).

They also show that DNase, besides some other possibledefenses, is an effective biochemical barrier to exogenousDNA in mammals against w'hich normal immunologicprocesses by antibody induction are not operating. Attempts to break this barrier will be feasible either throughprotection of substrates by complex formation withdyes or proteins or through use of natural DNase inhibitors(5, 15).

ACKNOWLEDGMENTS

We want to thank Dr. J. Huppert and I)r. J. P. Changeux forfruitful discussions and l)r. B. Bases for his help in translatingthis manuscript.

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2. Burton, K. A Study of the Conditions and Mechanism of the

4.

0.000.15 0.20

DNase ActivityKW

lrtsolDNA(,ug/@,)

I'@ DNA I@‘@DNAMG. I t

S %I

II

I

75

50

25

I ‘a

‘• I.1 ‘a

S II ‘a

I ‘a

0/ SSS

_0 50 160 I@0 200 time (mm)

CHART 7. Comparative study of plasma concentration of insol-DNA after i.p.injection of l)NA (Sample c) and injection of the DNA-MG complex into Swiss mice.

Vol. 25, July 1965

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GOSSE et al.—Initial Degradation of DNA 883

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