A circular channel crucible oscillating viscometer

./.

,i!O/.

Rid.

(I!%])

A Circular

147. .!i()l-r,21

Channel

Crucible

Oscillating

Viscometer

Detection of DNA Damage Induced in Viva by Exceedingly Small Doses of Dimethylnitrosamine SII,VI~

I’ARODI~~. MAIWZIO

PIA (‘.AHIA~~, ANTONIETTA TANINGHER~.

MARTELLI~

RENATA FIN~LLO’

MAVRO PALA] and WAI,TER

(:IARETT?

‘Isfituto Scirnti$co prr lo Studio P la Cura dri Tumori Ikpartmrnt of O~rcology, Univwsity of Genoa, Italy 21jppa,rtment of Pharmacology, Univrrsity of Gnoa. Italy 3Drpartmrnt of Experimental Physics. Politecnico of Turin, Italy (Kpwiwd

21 July

1980. a&

irr rwiwd

form

17 Ikcm,brr

1980)

X ne~v oscillating crucible viscometer. having a U-shapd circular channel, is drscrilwd. The damping coefficient 6 is lowered by an increase of the viscosity 7. The instrument described here allows t,he solution to come in contact with inert plastic only. At all steps of its preparation and during viscosity measurements. giant DNA from rat liver nuclei was maintained at shear stresses around 10e4 dynes cm-‘. Viscosity was studied as a function of surface tension, DNA concentration and shear stress. It was found that under our experimental conditions it was possible to obtain meaningful values for reduced viscosity, q,,,+ practically identical to intrinsic viscosity 171. Rat liver nuclei are incubated in an alkaline lysing solution (pH 12.5: 22°C): they are lysed immediately and the released DNA starts to uncoil. The viscosity of solutions of this giant DNA increases very slowly with time. reaching a maximum only after about ten hours. The process was accelerated by single-stranded breaks arising from methylation of DrSA in ciao with dimethylnitrosamine. It was found that’ the time of DNA disentanglement, was sensitive to an exceedingly small number of breaks. We think that we were able to measure molecular weights around the length of the single strand of an average chromosome (M, -5 x IO”). An empirical relation between molecular weight and reduced viscosity after complete disentanglement was also rstablished. as a linear log-log plot, covering a molecular weight range between lo* and 2.5 x IO”. It is suggested that the viscosimetric evaluation of DNA disentanglement is probably the most sensitive method for studying DNA damage induced “i,l viuo” by chemical carcinogens.

1. Introduction This work has two main focuses of interest. For t,he first time we describe in a fulllength paper the adaptation of a circular channel crucible oscillating viscometer t,o the biological problems of very high molecular weight, DNA. This instrument is t Author to whom requests for reprints shoultl be sent, at Istituto ritsi ‘I’umori. Viale Henetietto SV. 10. I-l613L’ (ienova, Italy.

Scientificw per lo Studio e la Cura

502

S. PAKODI

ET .4L.

entirely different in design and principle from the well-known concentric cylinder viscometers of the Zimm-Crothers type (Zimm & Crothers, 1962; Gill & Thompson, 1967: Bowen & Zimm, 1979). The second focus of interest of our work is the demonstration that with this instrument detection of exceedingly small levels of DNA damage (after treatment in viva with a chemical carcinogen) is possible, These levels can be extrapolated, as a first approximation, to molecular weights around 5 x 10”. This is in the range of the single strand of an average intact chromosome. There is an overwhelming consensus in considering DNA damage as the initial step of carcinogenic processes induced by the large majority of chemical and physical agents (Cleaver, 1968, Sarma et al.. 1975; Stich et al., 1976; Petzold & methods for evaluating low levels of DNA Swenberg. 1978). In consequence, fragmentation after treatment in vivo with a chemical agent are of potential significance also with respect to their possible practical application as short-term prescreening tests of carcinogenesis. In this respect sensitivity to very low levels of DNA damage is an essential point because, after an acute or subacute treatment itt vivo with a chemical carcinogen, the number of breaks from labilized points in DNA could be exceedingly small. To our knowledge, the results presented here have probably the greatest level of sensitivity achieved so far. The main reasons that explain the extreme sensitivity of our viscometer are probably three: (1) while DNA from mammalian tissues can be lysed directly in our U-shaped circular channel without any significant DNA shearing, at the same time a sufficiently homogeneous DNA distribution in the alkaline solution can be achieved, allowing for consistent and repeatable results; (2) the working shearing stress range is very small for our viscometer : lop4 to 10 x 10e4 dynes cm -2 ; it is of the same order as the lowest shear stresses that can be obtained with the ZimmCrothers type of viscometers (Klotz & Zimm. 19726), (and it could easily be reduced further); (3) the recipe for preparation of our liver nuclei is probably almost optimal for achieving the double goal of leaving a negligible amount of protein and other macromolecules as contributors to the viscosity of the lysed material, and at the same time insuring intactness of giant DNA. Maximum sensitivity to the lowest levels of DNA damage was shown by measuring DNA disentanglement rates, not’ the levels of the maximum viscosities observed after completion of DNA disentanglement.

2. Brief Description

of Apparatus

and Theory

The apparatus is shown in Figures 1 and 2. Basically, the viscometer consists of an oscillating crucible suspended by a wire and containing the liquid under study in a half-filled, U-shaped circular channel. The parameter measured is the harmonic damped oscillation of the crucible. This damping depends on the frictional torque exerted by the liquid on the internal walls of the circular channel. The oscillations which gives a starting torsion to the are started by a rotating device, nickel/chromium wire from which the crucible is suspended. The damped oscillation of the crucible is registered by a spot follower. whose mobile equipment receives the light beam reflected by a mirror attached to the axis of the doughnut (Fig. 1). The doughnut is made of Perspex (Montedison, Italy), which allows the use of strong

VISCOSITY

AND

DISENTANGLEMENT

OF LIVER

DNA

503

Fro. 1. Schematic diagram of the viscometer: A, starter; B, nickel/chromium wire; C, mirror; D-D,. resistance thermometer; E, t,hermistor with stirrer; F, oscillating crucible; G, ring cover; H. air bath; I. water bath: L, screw jack: M, temperature monitor: N, inertial rings.

acidic and basic solutions, and temperature variations up to 80°C. Figure 2 shows a cross-se&ion of the crucible and its dimensions; the inner channel was made with an accuracy of 10m2 mm. The initial and maximum angle of oscillation is - 2”. The radius of the nickel/chromium wire is r = @15 mm, its length 1 = 600 mm, and its elastic torsion constant, measured with the use of calibrated rings. is li = 8.46 x 10’ 1 +O.Ol dynes cmP2. The period of oscillation of the empty crucible. obtained from an experimentally measured total moment of inertia I = 17960 + 6 g cm2, 1s ’ T, = 24.51 seconds for the wire used for these calibrations. Changing the wire (every few months) generates small, but not negligible, changes in To, at the

:::: k 0

: IO mm

;+45.od: id+---53.5 :o-----

+ 20

: ; j

46.5

hi ,I

63-5

i i

: ; :

Vi

FIG:. 2. (‘rowsection ofthe oscillating rrwible with the half-filled IT-shaped circular c,hannel. The radii at different plane se&ions are given in mm. h is the height of the liquid in the circular c,hannel. K = 450 mm is the radius of the torus and n = 3.5 nun is the inner ratliw of the cahannel.

level

of the t.enth of a second of the period length. 7’, of the wire used in t’ht% with dimethylnitrosamine was 2420 seconds. A copper container. complet’ely immersed in a waterbath. isolates a region of constant temperature. where the crucible oscillates. The temperature is regulated with an accuracy of +@05 deg. C. Two resistance thermometers, placed in the proximity of the doughnut. monitor temperature variat,ions during the experiments (Fig. 1). The basics of the theory of the instrument have been described, Gallina rt a/. (1971) demonstrated that for a toroidal channel the theory is able to relate the observed damping coefficient S (logarithmic decrement of oscillations) to the coefficient of viscosity of the liquid 7. The fundamental result of the theory is:

experiments

l(S-S,)Zf 4da2R3

P

(1)

where I is the moment of inertia of the oscillating system : T and To are the periods with and without liquid, respectively; S and So the logarithmic decrements wit,h and without liquid, respectively; p is the density of the liquid; v the viscosity of the liquid : a the inner radius of the channel ; R the radius of the torus (Fig. 2) ; q the dimensionless parameter given by a(2np/TT)f: and (J,, Cl,. G3 are the universal functions of q, the tabulation for which was given by (:allina rt al. (1971).

VISCOSITY

AND

DISENTANGLEMENT

OF LJVEli

DNA

,505

Using our experimental conditions 6, is 0.68 x 10e4 s- ‘, the range of variation of (S-So of 0.9?; (w/v) Sarcosyl in water) (S-S,) is between 527 x 10m4 s-l and 3.09 x 1OY4 s- ’ (6 - 6, of 0.90/o Sarcosyl +300/o (w/v) sucrose, in water) and the Under these conditions range of T between 24.29 and 24.34 seconds, respectively. the range of q is between 1.82 and 1.09. For our values of 6 and a2/R2, the second and the third term of the right side of equation (1) are negligible. and the only important contribution comes from the first term. The graphical representation of (:, as function of q is shown in Figure 2 of Gallina et al. (1971). Our range of q, mcntion~~d above. is in the left part of the curve. In this part of the curve t,he variat,ion of (a-6,) for different values of 17is maximum and results in a higher sensitivity of the instrument. Equation (1) gives absolute values of 7 only for a caompletely filled toroidal channel and for measurements made under vacuum. These two conditions, and in particular the first and most important one: are very difficult to satisfy without shearing of high molecular weight DNA. Even if we have not yet adapted the theory for a half-filled U-shaped circular channel in order to obtain absolute values of 7 directly from (S-S,), it was easy to standardize our \-iscomc%rr against sucrose solutions of different concentrations.

3. Materials

and Methods

(a) Chemicals Heparin was purchased from Vistar, Como, Italy, and thiopcntal (trade name : Pentothal) from Farmitalia, Milan, Italy ; (ethylenedinitrilo).tetraacetic acid disodium salt (Na, EDTA), sucrose, 20% tetraethyl ammonium hydroxide, dimethylnitrosamine and bovine serum albumin were obtained from E. Merck, Darmstadt, Germany; N’-lauroyl sarcosine sodium salt (Sarcosyl) from Sigma Chemical Co., St Louis, MO., U.S.A.; trypsin I : 250 was bought from Difco, Detroit, Mich., U.S.A. ; Triton X-100 from Kodak, Rochester. 1T.S.A.: and 3.5diamino benzoic acid dihydrochloride from Fluka A. (i., Bucks, Switzerland. (b) Preparatio?t

of l&r

Euclei

For evaluating the viscosity of high molecular weight unsheared DNA, purified live1 nuclei were prepared as follows. Sprague Dawley male rats 5 to 6 weeks old were used in all experiments. At this age rat liver cells are mainly diploid. Rats were fasted for 24 h before sacrifice. They were heparinized with @l ml 5%& (w/v) heparin/kg and anaesthetized with 50 mg thiopental/kg, both injected intraperitoneally. After the rat’s abdomen was opened and the portal vein carefully isolated, the liver was perfused for about 5 min with 50 ml of OTV& (w/v) NaCl at - 2O”C, followed by 30 ml of trypsin-EDTA solution at 37°C (0.04%) (w/v) trypsin, 002% (w/v) Na,EDTA). The venue hepaticae were opened and a further PO ml of trypsin-EDTA solution injected into the cannula. The liver was then excised and minced with scissors in 30 ml of ice-cold dissociation medium (0075 iv-NaCl, 0024 MNa,EDTA, pH 7.5). All subsequent steps were carried out at - 4°C. A rough liver cell suspension was obtained with 3 strokes of the pestle of a loose-fitting homogenizer, and was filtered through a 60 mesh stainless steel sieve. Cells were collected from the filtrate by centrifuging for 2 min at - 15Og. About 2 ml of the pellet was resuspended in 4 ml of dissociation medium, and 4 ml of water added to haemolyze erythrocytes by osmotic shock. The suspension was centrifuged again for 2 min at - 150 g. This step was repeated to avoid incomplete haemolysis. The pellet was resuspended in approximately 7 ml of dissociation medium including @75% (v/v) Triton X-100 and incubated for 4 min. The suspension was centrifuged for 2 min at - 15Og. This step was repeated with a 30s incubation, once or twice, until the pellet had changed in colour from a very pale brown to completely white. Thr

MM

S. PAl(

E7’.-IL

pellet was washed once in 5 ml of dissociation medium and resuspended in the same medium to the approximate concentration needed for a given experiment. The DNA. RNA and protein content in our nuclear preparations, after the last purification step, with nuclei suspended in dissociation medium, were assayed by classic calorimetric methods. DNA was measured by the diphenylamine reaction method according to Burton (1956) ; RNA was measured by t’he orcinol method according t,o Ceriotti (1955). I’rot,ein was assayed by a modification of the Lowry method (Hartree, 1972). The range of ratios bv weight among DNA, RNA and protein for our nuclear preparations was found to be I.0 : 0% to 0.8 : 6 to 12. At, different times, .5 different preparations were examined and found to vary only within these ranges. (c) Twatment

of animals

with

dimethylnitrosamivw

Sprague Dawley male rats 5 to 6 weeks old were treated intraperitoneally with single doses of DMNAt dissolved in @9% (w/v) NaCl (pH 7.0; 001 ml/g body weight). Controls received the same volume of physiological saline. Animals were sacrificed always 4 h after treatment,. For viscosimetric experiments, doses spanned the range from 0.022 to 5.40 mg/‘kg body weight. For each dosage level, the experiments were repeated 4 to 5 times. For alkaline elution experiments, doses spanned the range from 1.25 to 20 mg/kg. Each dosage was repeat,ed 5 t’o 6 t,imes. (d) lTiscosimutric

assay

The circular channel of the crucible was partially tilled with a constant liquid volume of 10 ml. All the experiments were run at 22°C. The standardization of the relationship between logarithmic damping and viscosity was performed with solutions of sucrose in water (including @go/o (w/v) Sarcosyl), at concentrations from Ooi, to 4%, as shown in Figure 3. As can be seen from the graph in Fig. 2 of Gallina Pt al. (1971), we would expect that (6 -8,)/p (which to a good approximation is directly proportional to 0, (q) according to eqn (1)) should be linearly correlated with Q for values of q bet,ween 0% and 1.5 ; for q > 1.5 (6 - 8,)/p should start to increase less rapidly than 9. As shown in Fig. 3, both expectations were found to be confirmed to a satisfactory degree. This fact indicates that our working conditions with a Ii-shaped circular channel remain relatively close to the theoretical conditions described for a toroidal channel. When (S-6,) for the same standard solution of water +@9u/, Sarcosyl and 100, or ZO(s,, sucrose, was evaluated on different days, i andj, (S - So)i/(S - S,)j was usually @99 to I.01 and never exceeded @98 to 1.02, even for 300/,, 35”i, or 4OyA sucrose. This precision was found to be adequate for our biological investigation. Probably it could have been even better if damping could have been measured automatically with an electronic device instead of manually from the graph of the spot follower. For lysing the nuclei in the circular channel of the viscometer, 2 ml of lysing solution A (38 mM-NaOH, 20 mM-Na,EDTA, 2 iv-NaCl, 2.20,; Sarcosyl, pH 9.1) were layered on the bottom of the circular channel, and 1 ml of nuclear suspension was added gently drop by drop. With our experimental conditions the number of drops is about 30, and special care is taken in order to insure that the drops are evenly and symmetrically distributed around the circular channel. To the meniscus of this solution was added gently 1 ml of lysing solution A, followed by 5 ml of alkaline solution B (8 mMNa,EDTA, 383 mM-TEA-OH, pH 13.4) and, finally, 1 ml of solution A. The final pH was 125. The viscometer was filled on a balance to a final weight of 1040 g, in order to insure filling to constant volume. The height of the liquid in the circular channel was only 6 mm ; thus the addition of the different solutions, drop by drop, should insure sufficient mixing along the vertical axis. On the other hand, heterogeneous distribution of DNA along the circular channel is not likely to be corrected by mixing. Therefore, we carefully distributed the drops of the nuclear suspension symmetrically, as described above. From the results t Abbreviations used: DMNA, tlimethylnitrosaminc; ‘I’EA-OH, tetraethyl I)ARA. 3,5-&amino benzoir acid dihydrochloricle; S.E strtndani error.

ammonium

hyclroxide:

i’ISCOSlT\i

AND

I)ISEN’l’A~GLEMEN’I’

OF LIVER

mi

I)NA

Sucrose concn W, w/v) 40

35

30 I

20 I

IO I

0 I

FH:. 3. Standardization curve. relating Q. where 4 = n (%~p/$/‘)~ to experimental inrrrasinp concentrat,ions of suc~rose. at WCI and pH 7.

(6-&,)/p.

fhr

obtained. it seems that the distribution was sufficiently homogeneous for our needs. At this point the circular channel was sealed with its cover, the doughnut was suspended from its nickel/chromium wire and the measurement of the damped oscillation was started after about 20 min (thr time required for temperature equilibration). Room temperature w-as maintained at about 21 “C. In order to verify that in our experimental conditions viscosity was essentially generated only by high molecular weight DNA, two different controls WRPV performed. (I ) Solutions of albumin, in the same final alkaline lysing solution used for DNA. a,t concentrations up to 10 times higher than the concentrations of proteins in our nuclear preparations. were measured for their viscosity. It was found that the viscosity of these preparations was practically indistinguishable from the viscosity of the final alkaline lysing solution. (2) After many viscosimetric experiments (approximately 1 out of 2 experiments), the lysed material was sheared by passing it 5 times forcefully through a 25gauge needle. 111 all cases. after shearing, the viscosity was equal or extremely close to the viscosity of the alkaline Ipsing solution.

(TV).-I ssay of 0~V-4 concentration

for viscor9imetric

measuv-emrnts

Nucleic acids were extracted essentially according to Schneider et al. (1950) with minor modifications. Briefly, the method was as follows : alkaline lysed material recovered after the viscometry experiments was diluted 1 : 4, precipitated with 10% (w/v) cold trichloroacetic acid and bovine serum albumin carrier (@8 mg/ml). The pellet was washed with 80% (v/v) et,hanol. DNA was hydrolysed in 0.7 M-perchloric acid for 30 min at 70°C and separated from insoluble material by centrifugation. This step was repeated twice. The DNA assay was essentially according to Kissane & Robins (1958)> with minor modifications. Briefly, t,hr method was as follows: the supernatant was diluted 3 : -5 and buffered at pH ‘7 with Na,CO, and Tris. HCI buffer. One volume of this solution was mixed with 1 vol. 1% MDARA in deionized water, final pH 1. The mixture was incubated for 30 min at 70°C. After 18

5OH

S. PAKODI

E’/’ AL.

cooling. it was diluted 1 :lO in 0.9 iv-perchloric acid. The fluorescence \vas read at SO nm with an excitation wavelength of 400 nm in a Perkin-Elmer 3000 fluorescence spectrometer. Assays were run in quadruplicate with duplicate standards of DNA at 2 different concentrations and duplicate blanks. each time.

The alkaline elution was done essentially according to Kohn r/ al. (1976). with minor modifications (I’arodi rl al., 1978). Approximately lo6 liver cells Were loaded on a Milliporr filter (mixed esters of cellulose. 2.5 mm diam.. 5 pm pore size), and washed Gth cold Merchant’s solution (0.14 iv-Nacl, 1.47 mM-KH2Ro,, 2.7 mM-KCl. 8.1 mM-xa,HP(j,, 053 mM-Na,EDTA, pH 7.4). The cells were lysed on the filter at room temperature with 4.5 ml of Iysing solution (2 M-NaCl, 20 mM-Na,EDTA. 0.2’?,, Sarcosyl, pH IO) and the filter was washed with 20 mM-Na,EDTA (pH 10). Single-stranded DNA was eluted in the dark with 20 ml of eluting solution (60 mM-TEA-OH. 20 mM-Na,EDTA, pH 12.3), at a pump speed of 0.2 ml/min, and 10 fractions were collected. At t.he end of elution the filter was broken up in d ml of eluting solution. The eluted fractions and the filter \vere assayed for DNA content, by the follovving modification of the microfluorometrir terhnique of Kissane Sr Robins (1968). To each 1 ml sample of the collected fractions. @I ml of bovine serum albumin (2 mg/ml). followed by 0.1 ml of lOOo,b (w/v) t,richloroacetic acid was added. The samples were then refrigerated for several hours and the precipit,ated DNA was pelleted by centrifuging at 17SOg for 15 min at 4°C. Supernatants were decanted carefully and I ml of 80% (v/v) ethanol was layered on each pellet. After a 2nd cantrifugation, the pellets were air-dried for 90 min at 50°C. Then 30 ~1 of a 40’:” (W/V) aqueous solution of DABA was added to each sample and the tubes were incubated for 30 min at 70°C. After cooling, 1.5 ml of 0.9 Mperchlorie acid was added t.o each tube. The fiuorescence was read as described in section (e), above. Blank readings jvere made from tubes containing 1 ml of eluting solution subjected to the same proredure.

4. Results (a) DNA

dise~itnnqlemrn

t

time in alkaii within the The apparent value of qrrxi as a function of incubation circular channel of the viscometer, for a typical control experiment and for nuclei from a rat treated with 0067 mg of DMNA per kg body weight four hours before sacrifice, is shown in Figure 4. It t,ook more than eight hours to reach maximum viscosity for liver DNA from the control rat and about 3.7 hours to reach two-thirds of maximum viscosity. In all six control experiments, the time required to reach maximum viscosity was between eight and 12 hours. For liver DNA from the treated rat. the time required to reach maximum viscosity was about four hours and two-thirds of maximum viscosity was reached in about l-2 hours. In all five to reach maximum experiments performed at this dosage, the time required viscosity was between two and six hours. As shown in Figure 4, after 24 hours or longer, some effect of degradation of phosphodiester bridges in alkali repeatedly became evident ; this phenomenon is not analysed further in this paper. In Figure 5 we show the results of four typical experiments. one for each dosage level, where the ordinate expresses v& as a fraction of maximum 7Rd at the plateau level. It was provisionally assumed that the slow increase of q,wl to a plateau level would mainly reflect, as a first approximation, the phenomenon of disentanglement of the two DNA strands.

YISCOSI’I’Y

AND

DISENTANULEMEN’I’

OF

LIVER

DNA

509

N b

x

4-

o-o-o O-O-.

0

I

2

3

4

5

6

7

Time(h) time in alkali for a typical FIN:. 4. Apparent values of reduced viscosity, ?wa, as function of incubation control experiment (0) and for nuclei from a rat treated with @067 mg DMNAjkg 4 h before sacrifice (0). Asterisks and arrows indicate the time (in h) required t,o reach a/3 of maximum viscosity.

Time(h) FIN:. 5. Ratio time. A typical DMNA/kg (A);

of reduced viscosity, ‘I~,, to reduced viscosity experiment is shown for each dose. Control 020 mg DMNA/kg (m).

at plateau (0); 0922

level, (o~),,,&~, plotted against mg DMNAjkg (a); 0.067 mg

In this context the mathematical treatment of the kinetics of DNA strand separations in alkali given by Ridberg et al. (1975) seemed convincing to us, and we assumed as a working approximation that 7Pd reflects the fraction of disentangled DNA. According to this line of reasoning, K, the rate constant of the relative

,510

S. PAROD

E’/‘iiL. Time (h)

FIG. ti. Kinetics of DSA disentanglement. Transformation of the curves of Fig. 5, according to eqn (2). The abscissa is proportional to t”‘67. K = -0.60 for the control experiment (0); K = - 190 fbr 0.022 mg DMNA/kg (0); K = -1.61 for @Of.?7mg DMNA/kg (A); K = -396 for 0.20 mg DMNAjkg (m).

increase

of7]rrc,, could be described

by the following

equation

:

K=h(I-=&“l’.

(2)

where (v~)~ is the apparent vEd at time i and (T~~),,,~~ is yWd at plateau level. If equation (2) describes, to a reasonable approximation, the increase of TRd with time, t,hen the curves shown in Figure 5 should display a reasonable fit with a straight line in a graph where on the abscissa we have te6’ and on the ordinate In (l~mJ(~~,&,ax). The results of this transformation are shown in Figure 6. The line of reasoning adopted seems to assure a sufficient approximation for the operational purpose of obtaining an objective K value from each curve of disentanglement. The average value for K (+s.E.) obtained for each DMNA dose is shown in Table 1. Experiments were run at least in quadruplicate.

$1 Dependencesf’ hpdLnr on

dimPthy1,nitrosamin.e

dosage

4s shown in Figure 4, after progressively shorter times for increasing DMNA dosages, qred reaches a plateau level. followed by a slow decay. For control DNA, this slow decay of viscosity is due to the instability of the phosphodiester bridges (Shooter, 1976). For DNA treated with DMNA or similar alkylating agents (under our experimental conditions), apurinic sites are the major cause of breaks in alkali

VISCOSITY

AND

DISENTANGLEMENT TABLE

Relation

OF LIVER

DNA

do.sr

and

1

between dimethyln,itrosamine disentanglement rate

DMNA dose (mg/kg)

-51 1

K+SE. -@60f0Q60

002“

- 1 .o(i * ofL54

0.06;

- 1.47 + 0.074 -2.71 kO.319 Not mertiiurable

0~20 o+io - 540

(C-4)

In all cases the difference between two consecautive dosage levels was statistically significant (I’ < @05. &tailed) according to the R’ilroxon 2 samples test (Siegel, 1956).

and their decay is practically complete after four hours (Peterson et al., 1974). In consequence, (q,Pd)maxfor DMNA doses of O-20 mg/kg and above was always taken after four hours of alkaline incubation. For the lowest DMNA dosages and control plateau level was DNA (~lredmax was taken at later times, as soon as a maximum reached. For each single experiment (qredjmax was averaged over eight to ten measurements taken in a time interval of about three hours around the proper time. The observed relationship between (T~~),,,&~and DMNA dosage is shown in Figure 7. Even if experiments were run at least in quadruplicate and controls in sextuplicate. the standard error was very large for the two lowest dosages and control level. Differences between two consecutive levels of DMNA doses were statistically significant only above 0.20 mg/kg.

(c) Comparison

with sensitivity e&ion method

of the alkaline

To give an idea of the extremely high level of sensitivity that could be reached by measuring the rate of DNA disentanglement with our viscometer, results typically obtained with the alkaline elution technique: one of the techniques considered at present most sensitive in evaluating low levels of DNA damage, are shown in Figure 8. The standardization of the alkaline elution technique in terms of breaks per lo9 daltons is referred to experiments with X-rays reported recently by our group (Brambilla et al., 1979), using exactly the same technique, which in our laboratory has given essentially identical results for the same treatments. (d) Effect of surface tension on viscosity measuremen& In order to obtain rapid and complete lysis and solubilization of liver nuclei, a very high ( -0.9V0) concentration of Sarcosyl was present in the final alkaline lysing

S. PAKOI)I

lr“/’ ,

.dL.

r

[DMNA]

(mg/kg)

FIG. 7. Dependence of reduced viscosity at plateau level. (~n,c,)ma.. on DMNA dosage. L)ifferenw between 2 consecutive dosage levels statistidly not significant (I’ > 0.10. awording to Wilcoxon 2 samples test (Siegel. l!XX): (--)pp>oa5.

(. .) P-tailed).

3.6

II -

9-

7-

5-

3-

r’ IL 0 [DMNA]

FIG. 8. Initial were c~aldated

rate K (ml-‘) from previous

(mg/kg)

ofelution of DNA for different DMNA computations (Brambilla et al.. 1979).

dosages.

Breaks

per IO9 tlaltons

VISCOSITY

AND

DISEN’I’ANCLEMEN’I’

OF LIVEK

I)NA

,513

6.0

t

-m-a\

.-.

-m-

...

Plc:. 9. E:ftc? of’ surface tension. (&SO) as function of’ increasing Sarc*osyl concentrations: Sarcosyl+3W, suwose, pH 7; q . Sarcosyl in alkaline Iysinp solution. pH 12.5.

a.

This high concentration of Sarcosyl should be also more than adequate t’o make the effect of surface tension negligible on our viscosity measurements. This possible effect, was explored by adding increasing concentrations of Sarcosyl (up t.o l2”/b) both to our final lysing alkaline solution without Sarcosyl and to a solution of 3O”/b sucrose at pH 7. As shown in Figure 9, for the neutral sucrose solution (6 - 6,) increased up to a 0.20/O concentration of Sarcosyl, suggesting an apparent decrease of viscosity due to reduced surface tension. Above 0.20/b Sarcosyl, (6 - 6,) decreased regularly. as would be expected merely from the increasing contribution to viscosity of Sarcosyl itself. For the alkaline solution, not even the initial effect of surface tension at low concentrations of Sarcos.vl could be observed, perhaps because TEA-OH (Eel9 M) and NaCl (2.0-S M) already induce a large reduction of surfa,ce tension. In conclusion, using our experimental conditions surface tension was probably a negligible factor.

solut~ion.

(e) Effwt

of

DSA concentration

(c) on QJC

The effect of DNA concentration, c, on the value of qrd was explored systematically at a dosage of 0.60 mg of DMNA per kg for a range of concentrations between 10 and 60 pg/ml. The results obtained are shown in Figure 10. It is evident that in our alkaline conditions at high ionic strength, and for the range of

I.01

' IO

I 20

I 30

I 40

I 50

I 60

DNA concn (g/d11 x IO4

VI<:. IO. tletluwvl viswsit~y, 7 m,,plotted against I)NA conventration. a shear stress of’ I 7 x 10e4 ti,vnes vm-*.

The viscosities awe measured at

concentrations explored. there was almost no concentration dependence of 7n.d. Each point represents a different animal treated with the same dosage. At different, doses of DMNA we did not make a systematic exploration of dependence of 7rrd on c : however. the average DNA concentration of our preparations is - 40 pg of DNA per ml and a,lmost’ all samples were in the range of 20 to 80 p-g of DNA per ml. The general trend for all DMNA dosages; including control samples, was the same ; there around was practically no dependence of 7d on c. For DNA concentrations than the 40 k&ml. our rllpd value was much less dependent on DNA concentration viscosity of native bacteriophage T2 DNA (at neutral pH, 0.18 M-Nit+ : Brothers 8: Zimm. 1965). However. for the same contour length, alkaline single strands at - 0.9 M-Naf (our experimental condition) are much more compact and less viscous (- I/7 that for the above native condition); moreover: 7Jc was only negligibly dependent on c in the latter condition (Rosenberg & Studier. 1969).

The exact estimation in our U-shaped circular channel of the rate of shear strain or gradient in fluid velocity @) is difficult. We used a simple but very approximate formula. The central layer of liquid, which is at a distance a from the walls, was assumed stationary during the harmonic oscillations of the crucible. This is obviously much worse than the real situation, because the movement of the liquid is partially coupled to the movement of the crucible. According to this unfavourable hypothesis, for a shear strain defined as AxlAy, Ax could be made equal t,o 2nR9”/360” (with 9” = 2” for an initial hemioscillation of maximum amplitude), then Az/Ay = (2nR9”/360” x n) and /3 z [(2~&9”/360” x a) x (l/fi)] ; 7) x ,B is the shear stress.

VISCOSITY

AND

DISEN’I’ANGLEMEN’I’

OF

LIVER

DNA

,515

N

h x

2.

.

l .

l

.

.

-

.

.*

F 2 \” 5 F

t-

0

I

I

I

I

I

0.1

0.2

0.3

0.4

0.5

Shear stress (dynes/cmz) Frc:. 11 I)epmdenw treatecl rats

on shear of the retluced viscosity.

~Jc,

I

I

0.6

0.7

x IO3 of liver

DNA

from

DMNA

(060 mg,‘kp)-

In our experimental conditions the shear stress decreases for decreasing values of 9”. C’onsequent,ly. the dependence of 7~~~on shear stress can be easily studied in the same sample, approximately for a range between 0.3” and 2”. Figure 11 shows the typical result obtained for a +j value of 156 x 10e2 poise, a D?\‘A concentration of 32pg/ml andadosageof@60mgofDMNAperkg. Wewent toemphasize that, because of the features of our viscometer, in any single experiment T,,~ is always measured against decreasing 9” and, by consequence, decreasing shear stresses. For our operative range of shear stress (0.7 x 10e3 to 0.1 x 10m3 dynes cm-*) no effect, on T)~~was ever observed. This seems in agreement with a previous report (Crothers & Zimm. 1965), where it was shown that, for shear stresses around 10m3 dynes en-* and below. the dependence of vnd on shear stress is very small.

5. Discussion In this discussion we will touch upon three main points: (1) the merit, and (.onvenience of our type of viscometer: (2) our findings on viscosity after DNA disentanglement : (3) our findings on the phenomenon of DNA disentanglement in the range of extremely high molecular weights. With respect to the original (Zimm & Crothers, 1962) and improved (Gill & Thompson, 1967 : Bowen & Zimm. 1979) Zimm-Crothers-type rotating cylinder viscometers. our viscometer has the merit of being based on a different principle. Both instrumentation and theory are already well-established for the Zimm(‘rothers type of viscometers (above-mentioned references and Klotz & Zimm. 19720); however. our way of measuring viscosity is also founded on sound and precision of the theoretical work (Gallina it nl.. 1971). Th e sensitivity apparatus is already more than adequate for our biological measurements. Moreover. our instrument is a prot’otgpe: both the precision and convenience of measurement can be further improved. for instance by adopting a digital electronic system for rreording the damped harmonica oscillations. The shearing stress applied

516

S. PARODI

87’ dL

t’o naked DNA from lgsed nuclei is very low in all steps (including preparative steps) of the experiment,. The evaluation of viscosity can be carefully monitored from one moment to the next without any disturbance to the material contained in the circular channel ; therefore our instrument is extremely convenient for kinetic studies. Obviously we do not want to imply that the kinetics of disentanglement of t)his ext~rcmely high molecular weight DNA could not have been monitored with a %imn-O-others type of viscometer. Results published by Uhlenhopp & Zimm (1975) and Uhlenhopp (1975) suggest that their type of viscometer could also be csonvenient for this type of study. In the above-mentioned work disentanglement (unwinding) is also mentioned. but, no analysis of its kinetics and their usefulness for the estimation of extremely high molecular weights was given. Moreover. lysing intact, nuclei in a Zimm-Crothers-type of viscometer. wit,h a homogeneity sufficient to avoid convective movements of the solution and at) the same time without shearing effects on DXA during the preparative steps, perhaps could have presented some problems. To conclude the first point of our discussion. we can say that our viscometrr is an original and valid alt,ernative to the Zimm-&-others type. especially suitable for st,udging mammalian DNA of extremely high molecular weight. Concerning the results obtained with this viscometer and presented here; first’, we would like t,o comment briefly on three variables that can affect our viscosity measurements; namely. surface tension, DNA concentration and shearing stress. These parameters were examined, not in tine detail. but to an extent that a-e considered sufficient for the aims of the present investigation. Our observations suggest that under our experimental conditions. surface tension. DNA concentration and shearing stress are not significantly affecting the results. Even if we did not express our data in terms of 1~1, for all practical purposes our 71ra values are not much different, from the corresponding 171 values. If \ve mnv rxarninr the sensitivity of our viscosity measurements. when maximum plateau viscosity disentangled DNA instead of for a completely disentanglement itself is considered. we observe the following main facts. First of all. considering Figure 7 it appears that dispersion of the results around the average is rather large and a considerable overlap exists between consecutive doses of DMNA differing by a factor of three in concentration. It is evident that between two consecutive doses (a (~,
(DMSA

mg/kg):

(3) with 9S(& confidence

limits

of the

VISCOSITY

AND

DISEX’I’ANGLEMENT

OF LIVEI<

Molecular weight 2.5~10' I

2eJx10* I

2.5 x IO” I

-

IJNA

-

517

1

.

5 ,8 -

//../ .

5 .6 -

.

:: ,E 5, 4“u F’ 5 5 .2-

./ ./

5 ,o4 '87 /

.

4 ,6,

I

I

cs.40

I -2

I

I.80

I

I

I

0.20 0.06 0.60 [DMNA] (mg/kg)

I

1

-I

0

I

I

I 2 -In (DMNA doses)

0.022 I

I

3

4

1

FIG:. I?. Plot 01’ reduced viscosity at plateau level. (~wc,).I.,, as a func%ion of DMSA dosages and molecdar weight. The relation between DMNA dosages and molecular weight is according to eqn (4) of the t.rst.

slol)r 0.15 to 0.23. and r = 0.989. From Figure 8. -0.5 breaks per 1O9 daltons (above controls) seem to be generated by 2.5 mg of DMSA per kg. We ha\-cx to consider that the initial elution rate of the alkaline elution method evaluates only t’he breaks generat’ed between 0 and 18 minutes of elution (average incubation in alkali Y 9 min). For DSINA and other methylating agents, apurinic sites, generated from loss of S-7 methylguanine, are the major cause of breaks in alkali. Their half life was reported to be about 30 minutes at pH 12% and 20°C (Peterson d al.. 1974). From our own experiments (data not shown) under the exact conditions of our alkaline elution, the half life of alkali-labile sites was found to be about 35 minutes. The real number of breaks that will be developed by a longer incubation in alkali (like in our viscometry measurements) will be about six times the breaks determined from the initial elution rate, [S 2: l/( 1 -e -o’o2x9)]. where -0.02 was our experimental rate constant per minute of degradation. for alkali-labile sites in our experimental conditions, and 9 is the time of alkaline incubation in minutes. As a result we can assume approximately three breaks per lo9 daltons for 25 mg of DMNA per kg.

s. PAKODI

518

Assuming a linear relationship between be discussed later), and because M” = where M,

is the initial

equations

breaks and DMNA

(breaks

dosages (this point

will

1110 -___in Me) + 1’

size, then: M, =

From

ET’ ilL.

x.3 x 10s DMNA

(3) and (4) we obtained

(4)

dosage (mg/kg)’ the following

(7j,,Jmax = 35 x MZ’“.

empirical

formula:

(5)

The very small exponent of M, obviously directly reflects the relative insensitivity to variations in molecular weight of (T~),,,~~. Even for a completely random coil, a much larger exponent (- 0.5) would be expected (Tanford, 1967). At the very high ionic strength at which we worked, single-stranded DNA apparently collapses on itself toward a globular structure, perhaps because of complete shielding of all charged groups by mobile counterions. This hypothesis seems in agreement with the observation reported by Rosenberg & Studier (1969) : at alkaline pH and very high ionic strength (0.1 M-NaOH + 1 M-Nacl), [v 1 was approximately 30 times less for the same DNA than at a similar pH but at low ionic strength (low3 M-NaOH) (see Fig. 2 of Rosenberg & Studier, 1969). A further (probably more essential) reason for the small exponent of M, in equation (5) could be the following. The viscosity that we measure with our apparatus is clearly a dynamic viscosity, not the usual steady-state viscosity. Dynamic viscosity depends on frequency (Ferry, 1961), and its behaviour with molecular weight will not be the same as the usual steady-state intrinsic viscosity. Our viscosity measurements are made at one particular frequency, about 094 reciprocal seconds. This frequency is much faster than the reciprocal retardation times that Unlenhopp found with intact mouse DNA, which were 600 seconds or more (Uhlenhopp, 1975). From the relation between M and retardation time (T) reported by the same author (M = 2.72 x lo* 7@565),7 is already of the order of the period of our oscillation for M = IO9 to 2 x 109. From basic relations amongst dynamic viscosity, retardation time and frequency, of the type suggested in Appendix F of Ferry (1961), we would roughly expect that the exponent of M, in our equation (5) could very well be much closer to 05 below M = log, and perhaps even lower than 0.19 above M = 109. This subject is obviously only superficially touched here, and needs much further investigation. From a biological point of view. the most interesting part of our work is the finding on DNA disentanglement, and the first point that we would like to discuss in this respect is the limit of sensitivity that was reached with our method. Some extrapolation is possible by making reference to our alkaline elution data. Using molecular weight extrapolations referred to our previous estimates (2.5 mg DMNA/kg cz 3 breaks/lo’ daltons) and. if we make the simplest assumption of a linear extrapolation. 0.022 mg of DMNA per kg will correspond to a range of - 3 breaks per 10” daltons.

VISCOSITY

AND

DISENTANGLEMENT

OF LIVER

DNA

519

From the work of Pegg & Hui (1978) on DMNA adducts in rat liver DNA four hours after treatment, it seems that the number produced decreases rather linearly from the milligram level to doses hundreds of times lower. However, while S-7 methylguanine (which is by far the most abundant adduct) seems to decay at a of alkylation relatively constant and slow rate at all dosages, disappearance products like O-6 methylguanine is significantly faster at dosages of 0.01 to 050 mg/kg than at dosages of 1 to 20 mg/kg, and an intermediate step of this repair process should be generation of further apurinic sites. In consequence, we do not claim that a linear extrapolation is entirely correct, but it can be considered useful to give a first hint of the sensitivity of the method. The surprising conclusion seems to be that if the DNA content of a single diploid rat nucleus is assumed to be 2 7 x lo-‘2 grams, then the DNA content of an average chromosome will be 7 x lo-‘2 /40 2 1.75x lo-l3 to a grams, or N 10 l1 daltons , which is equivalent molecular weight of - 5 x 10” daltons for a single strand. In other words, it seems that our method is capable of detecting a size range around the size of the average chromosome. In addition to its sensitivity, DNA disentanglement has a further important advantage: DNA concentration measurement is not required for this parameter. which makes its measurement much more direct and practical than the measurement of (7red)max.Another corollary of our results is the following: having increased the sensitivity about 100 times with respect to alkaline sucrose gradient sedimentation, we can reject the hypothesis of a relatively fast formation in alkali of single-stranded subunits around lOa daltons as reported by Filippidis & Meneghini (1977). who suggested production of these subunits after an exposure of only five munites at 20°C to 0.15 M-NaOH. This observation seems extremely difficult to reconcile with our results: both for controls and the lowest DMNA dosages we should have always observed a completely disentangled DNA from the beginning of our experiments, without any possibility of discriminating the lowest dosages from control DNA. Our results strongly suggest that each single strand of the chromosome is probably continuous. Alkali-resistant linkers are not excluded (Hershey & Werner, 1976) and our instrument could be useful in the investigation of this point. after treatment of lysed nuclei with Pronase. The importance of measuring DNA damage in viva in a specific target organ has been repeatedly underlined. Metabolic mechanisms of the intact animal are often only poorly reproduced, and pharmacokinetic events (absorption, distribution and elimination) are t’otally absent in in vitro systems. Chronologically, the first practical approach in vivo was the evaluation of rat liver DNA sedimentation velocity in alkaline sucrose gradients (Cox et al., 1973; Damjanov et al., 1973). More recently, a procedure suggested by Kohn et al. (1976), measuring the rate of DNA alkaline elution through filters with calibrated pores, became common. DNA damage induced in vivo by many carcinogens has already been investigated by this technique (Parodi et al., 1978,198O: Petzold XI Swenberg, 1978). The limit of sensitivity of this method can be considered to be around 0.5 to 1.0 breaks per IO9 daltons. Unfortunately, for a significant number of carcinogens a brief treatment (even with maximum tolerated doses) generates only a minute number of DNA adducts. and therefore of DNA breaks (these are always a small portion of DNA adducts). This number is too small to be revealed within the above limit of

520

S. PAKOi)l

E7’rlL

sensitivity. In consequence. a method markedly more sensitive than alkaline elution could be wry useful in identifying the usually lower level of DNA damage induced, for instance. by some arylating agents (Janss & Ben, 1978) with respect to alkylating agents (Pegg & Hui, 1978). It \zns recently shown by our group. that for a family of 16 hydrazine derivatives, the evaluation of DNA damage in lung and liver DNA of mice (by the alkaline elution assay) was beyond doubt much better correlated with carcinogenic pot,ency in mice than the Ames test. At the same time. for the weakest carcinogenic hydrazine derivatives. the alkaline elution assay did not appear sufficiently sensitive (I’arodi et ul., 1981). In consequence, our instrument. and our approach to the study of DNA disentanglement. could have useful applications in this area. The authors are grateful t,o Dr E Patrone of the “(!entro Studi C’himicofisici Macromolecole Sintetiche e Naturali” of Genoa. for his helpful suggestions in discussing the viscosity. The authors thank Mr C:. relationships between dynamic and steady-state Accornero. technician of the Department of Pharmacology of (Genoa University. for the construct,ion of the viscometer apparatus. according to the project of one of the authors (W.C.). This work was supported by a grant from the Consiglio Nazionale delle Ricerche 78.028.56.96 and (IYoget,to Fillalizzat,o ‘Y‘orttrollo drlla Crewit.a Seoplastica” ( contracts: 78.02803.96). REFERENCES Bowen, B. CI. & Zimm, B. H. (1979). Biophys. Chem. 9, 133-136. Brambilla, G.. Cavanna, M., Carlo, I’., Finollo, R.. SciabB. L., Parodi, S. & Bolognesi, C. (1979). J. Cancer Res. Clin. Oncol. 94, 7--20. Burton, K. (1956). Biochem. J. 62, 315-323. Ceriot,ti, (:. (1955). J. Biol. Chem. 214, 59-70. Cleaver, J. E. (1968). Xaturr (London), 218. 652~656. (lox, R.. Damjanov, 1.. Abanobi, S. & Sarma, D. S. R. (1973). Cawrr Rus. 33, 2114-2121. Clrothers, D. M. & Zimm, B. H. (1965). J. Mol. Biol. 12, W-536. Damjanov, I.. Cox. R.. Sarma, D. S. R. & Farber. E. (1973). Cancer Res. 33, 2122~2128. Ferry, .J. D. (1961). T’iscoplastic Propwtirs of Polymrrs.
4629-4637. l’arotli, S.. Taningher,

M., Santi, L.. Cavanna. (1978). M&al. Kes. 54, 39WM5. Parodi. S.. Taningher, RI.. I’ala. M.. Brambilla. Health. 6, 167-174. l’arodi, S.. De Flora, S., Cavanna. M ., Pino. A.. (1981). Carlcer Rcs. 41. l’egg. -4. E. & Hui, (S. (1978). Biochrm. J. 173. Peterson. A. R., Bertram, .J. S. & Heidelberger,

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Sriab&,

L.. Mama.

G. & Clavanna,

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739-748. C. (1974). Cawrr

A. & Brambilla,

(:.

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Res. 34. 1592-1599.

(:.

VIS(‘OSI’I’Y

AND

DISENTANGLEMENT

OF LIVER

WI

DNA

I’etzold. (:. L. & Swenberg, J. A. (1978). Cancer Rrs. 38, 1589-1594. Ridberg, B. (1975). Radiat. Res. 61, 274-387. Rosenberg, A. H. & Studier, (1969). Biopolymera, 7, 765774. Sarma, D. S. R.. Rajalakshmi, S. & Farber, E. (1975). In Cancrr (Becker. F. F., ed.). vol. 1. pp. 235271. Plenum Press, New York and London. Schneider, W. C., Hogeboom, G. H. & Ross, H. E. (1950). J. Sat. Cancer Inst. 10. 9777983. Shooter. K. V. (1976). Chum. Biol. Id. 13. 151-163. Siegel. S. (1956). Son-parametric Statistics: ,for the Behacioral S&vu-rs (Mr(:raw, ed.). Hill Book Company, Inc., New York. Stich. H. F., San. R. H. C., Lam. I’. P. S., Koropatnick, D. J., Lo. L. W. & Laishes. B. A. Tvsta i/l Chemical Carcinogenesis (Montesano, R., Bar&h, H. & (1976). ln Screening Tomat,is. I,.. eds), vol. 12, pp. 617 -638, International Agency for Research on (Jancer. Lyon.

Tanford.

(1. (1967). Physical

Chemistry

of Macromoleculrs.

I’hlenhopp. E. L. (1975). Biophys. J. 15, 233-237. IThlenhopp. E. L. & Zimm, B. H. (1975). Biophys. %imm. H. H. & Crothers, D. M. (1962). Proc. Sat.

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J Wiley

$ Sons. Inc..

48. 905-911,