Shock Waves and Free Radicals

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MATERIAL &. METIIODS

Ll210 Cells ill yjtroThe permanently growing suspension cell .line LillO was c u l t u r ~ d f o ~ 14 days in ~ h e ahsence or presence of different concentratIons of a-tocopherol. V,tamln E was apphedas an emulsion of 5% a -tocopherol and 15% modified fish gelatine, or as purea-tocopherol, partially emulsified in the culture medium by sonication. Cells were

washed twice before treatment, so that only a-tocopherol incorporated in the cellmembrane could be effective. Shock waves were applied in an experimental Domierspark gap lithotripter XIA (generator voltage 18 kV, capacitor 80 nF). Water in thelithotripter bath (37"C) was partially degassed (-2 mg 0,11). After administration of

250 shock waves at a repetition frequency of I or 8 Hz, cell viability was determinedusing a cell counter and a double staining technique: cells that were able toenzymatically hydrolyze the membrane-permeable dye fluorescein-diacetate and exhlbita green fiuorescence of accumulated fiuorescein, were considered as viable, whereascells which cannot exclude the membran-impermeable dye propidium ioilide andexhibit a red fiuorescence are physiolgically damaged and must be regarded as dead.

The amount of viable and dead cells was determined using flow cytometry (4).Additionally, the growth rate of cells after shock wave treatment and untreated controlswere compared for at leasl9 days by seeding equal concentrations of cells and countingthem daily.

Rat El)'Ihrocytes a viva

Rats were kept for 6 weeks on a adequate or tocopherol-free diet (11). Aftertocopherol depletion of the rats, ascenained by an increased serum pyruvate kinaseactivity (11), blood was obtained by heart puncture and stabilized by citrate anddextrose. Blood samples of each animal were split for three treatments, and the cellswere washed with phosphate buffered saline for sbock wave treatment After treatmentwith 250 shock waves at a repetition frequency of I Hz, cells were centrifuged, thenumber of intact cells was determined in an electronic cell counter, and the amountof free haemoglobin in the supernatant was determined photometrically.

RESULTS

In all three experiments - the viability of Ll210 cells, the growth rate of Ll2lO cells,and the haemolysis of rat erythrocytes - a difference between low and bigh content ofa-tocopherol cells could be demonstrated after shock wave treatment

Ll210 cells supplemented with a-tocopherol were less damaged in botb acuteand long term investigations (table 1 &. fig. 1). The acute viability of Ll210 cellsexposed 10 250 shock waves after culture in the presence of a-Iocopherol revealed aprotective effect of the bighesl concentration (table I). The protection of Hr' Ma-Iocopherol was more pronounced for a sbock wave repetition frequency of 8 Hz,wbere more free radicals are produced than at 1 Hz [8,10).

. A more sensitive method Ihan the determination of acute cell death after shockwave ITeatmenl may be the investigation of long-Ierm effects, since cells injured by

s ~ o c k waves and free radicalswill be altered physiologically. As could be demonstratedWIth a-tocopherol pretreated Ll210 cells exposed 10 250 sbock waves, a concentration

616 Ultrasonics Intemational 93 Conference Proceedings



dependent protective effect is visible after a lag-phase of 72 b (fig. 1).

Erythrocytes from t ~ p h e r o l depleted rats showed an enhanced haemolysiscompared to adequate fed arumals (table 2).The percentage of geometrieally destroyedel)'throcytes after 250 shock waves, as determined with a cell counter, was 175 ± 1.4%for the control animals (mean ± s.d of n = 6 animals) versus 30 .6 ± 5.3% for thetocopherol-depleted rats (n = 5).

CONCLUSIONS

From our results, we conclude an involvement of cavitation-generated free radicals inshock wave induced cell damage in vitro. Scavengers of free radicals may be alsohelpful for clinieal use in vivo.

REFERENCES

(1)

(2)

(3)

(4)

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(6)

(7)

(8)

(9)

(10)

(11)

l..ingemu. J.E.... McAteer, Kempsollo SA and Evan. A.P. Bioe ffc:c:ts ofe.uracorporeal shock-wave lithotripsy. Strategy for research .. treatme.t lJrol. CIllt. North Am. (1988) 1l: 507-514Briinlmer, F., Brl uner, Th. and HUlser, D.P. Biological effects of shock waves World 1. Uro!'(1990) a 24-232 ..Russo, P., Stephenson, R.A., Mies, C . R . Heston, WD.W . Melamed, M.R . and Fair,W.R. High cDcrgy shock waves suppress tumor growth in vitro and in ";110 Ie UroJ. (1986) i l l 26-628Briimmer, F., Brenner, J., Brauner, Th. and HIUsct, D.P. Eifcd. of shock waves 00 suspended andimmobilized Lt210 ceUs Ultrasound Med . 8;01. (1989) 1l: 229-239Brauner, Tb ., BrUmmer, F. and HUIser, D.P. Histopathology of shock wave treated tumor c:eIl

suspensions and multiceU tumor spheroids Ultrasound Med. Bioi. (1989) .u 451460Gamblhler. S. aDd Delius, M. InflUCtlCC of dissolved ud free gases on iodine release and cellkiIliDg by <hock ....... in >iIro Ultrasound Mcd. BioL (1992) Ja 617-623

Steinbach, p..

Horstidter. F .. Nicolai, H . R6ssJer. W. and Wieland, W./II Yilro iDvestigations OQ

",Oular damage mdueed by hlgh energy shock waves U)Uasound Md. B;ol, (1992) Ja 691_Hengleio, A.. G u t i t l T ~ M. and Ulrich. R. Oxidatioo of iodide by the intense acoustic bursts ofan extracorporeallithotripter In t J, Radial, B;oI, (1988) 123-126Morgan. T.R . Laudone, V.p . Heston, W.D.W . Zc:itz. L and Fair. W.R. Free radical productionby high energy ,hock waves • Comparison with ionizing irradiation 1. Urol. (1988) JJ2. 186-189Suhr, D.. Briimmer. F. aDd HQlser, D.F. Cavitation-generated free radic:aJs dwing shock waveexposure: investigations with ceO-free solutions ud wspc:ndc:d"Us Uhrasoupd Med. Bioi. (1991)11761-768Weiser, H . Vecchi, M. and Sch1achlet. M. Sterc:oisomc:rs of .-toc:opheryl acetate. m.Simultaneous determination of resorption·gestation aDd myopathy in rats as a meaDS ofevaluatingbiopolency ratios of all-rae- and RRR-.-toc:opberyl acetate Internal, 1. Vit .NUll. Res. (1985) jS149-158

Table 1 Vital LI210 cells after 250 shock waves

pulse repetition frequencY

without tocopherol

supplemented with 10-2 M toe.

1 Hz

45.9 ± 4.4%

54.2 ± 1.8%

8Hz

51.7 ± 3.9%

66.4 ± 1.4%

Ultrasonics Intemationa( 93 Conference Proceedings 617



- e" -...

]

§C

-

107

10"

105

10'

b- i> toe. 10-2 M

t o e . 10-' M

D -O toe. 10-4 M

0 - 0 without toe.

10"+-- - - - . - - - - . - - - - - . - - - - - . - - - - ,

o 48 96 144 192 240

cultivation time / h

Fig. 1 Growth of L1210 cells after a 14-day incubation with different concentrationsof a-tocopherol. Upper curves: controls, lower curves: after 250 sbock waves.For further information see texl

Table 2 Destroyed erythrocytes (%) after 250 sbock waves

animaI # mean s.d.

1 172 3.02 19.1 3.S

control 3 IS.0 1.4group 4 IS2 1.0

5 14.9 3.66 175 25

7 322 2.7tocopberol- S 34.6 1.1depleted 9 32.9 1.6group 10 32.1 2.1

11 21.3 25

'This work was supported by F. Hoffmann-La Roche Ltd., BasIc, Switzerland.

618 Ultrasonics International 93 Conference Proceedings

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Shock Waves and Free Radicals