BAŞBAKANLIK
ATOM ENERrtSi KOMİSYONU
؟ E K M E C E N Ü K L E E R A R A Ş T IR M A
٧
E E Ğ İT İM M E R K E Z !
RAPOR No: ألأل
DETERMINATION OF THE T O X IC EFFECT
OF AET O N RAT EM B R Y O N IC CELLS
Ertan ÜÇER and E si. BÖLÜKBAŞI
STANBUL Ü N İV ER SİTESİ FEN FA K Ü LTES؛ MECMUASI
S eri B, C ilt X X X V II, SAYI 3 . 4
RKVUK DE IjA FA C U LTE d e s SCIENCES DE D’U N IV ERSITE D 'ISTA N B U L S é rie B. T om e X X X V II, EASC. 3 - 4
(A y r ı Baskt) 1973 (Re.print)
A £ T ’n؛n Sıçan Embriyo Hücreleri üzerindeki Toksik Tesirinin Tayini
Determination o f the T oxic Effect o f AET on Rat Embryonic Cells(*).
Ertan ÜÇER and Esin BÖLÜKBAÇI
(Çekmece Nuclear Research and Training Center, Istanbul, Turkey)
öıeV ٠. Bir radlasyotu koruyucusu olan AETnin, doku kültüründe yetiştirilen sıçan embriyo hücreleri üzerindeki, toksik tesiri tayin edilmiştir. \h ilâ 45 mM konsantrasyonlarda kullanılan AETnin, sıçan embriyo hücreleri İçin toksik olma- yan en yüksek dozunun ٦ ك١mM ١’e öldürücü dozunun da 45 mM olduğu tesbit edil- »niştir.
ﺃ ﺪ ﺳ ﻵ The toxic effect o f the radioprotector, A'ET for rat embryonic cells in tissue culture was investigated. Various concentrations o f AE Tfrom \b to 45 m M were employed in the experiments. As a result, tlie highest non-toxic and lethal doses o f A E T were found ٥؛ be 20 and 45 mM respectively.
The letlial and harmful effects of x-and gamma rays have been found to be reduced by the prior administration of ceitain substances. These chemical sub- stances are called radioprotectors. One of the common characteristics of radiop- rotectors is their necessary presence in the organism exposed to radiation.
Cystcin and cyanide were the first radioprotectors to be discovered in 1949 (PATT et a l, 1949; BACQ and ALEXANDER, 1966). Since then, numerous compounds have been reported to be radioprotective. In 1957, in the search for a new effective radioprotective agent, a sulphydryl compound, namely g-ami- noethylisothiouronium (AET) was discovered by DOHERTY and his friends.
(٠) This research work is a par، of ،he experimental series dealing with the Project "Some Internal and External Factors which Increase or Decrease Sensitivity Against Radiation" of Contract number 833/R1/RB and financed by the International Atomic Energy Agency in Vienna.
SERI B, CILT XXXVII SAYI 3 . 4 E. ÜÇER and E. BÖLÜKBAŞI
146
Ih is compound changes to mercaptoethyl guanidine (MEG) at neutral pH values, and in fact MEG is the essential protective molecule (BACQ and ALEXANDER 1966). The radioprotective effects of AET have been confirmed by other inves- tigators in several organisms also (LEONARD and MAISIN, 1964; NEWSOME and OVERMAN, 1964; MAISIN et ٠/., 1965; NEWSOME et al, 1967).
Since most of the radioprotectors are toxic for the organisms, the highest non-toxic concentration of AET was determined in tire experiments.
MATERIALS AND METHODS
Embryos of rats (Albino, Rattus norvegicus) reared under standard con- ditions in our laboratories were employed in the experiments. The pregnant rats were sacrificed under sterile conditions in the tissue culture laboratory. Primary cell cultures were prepared from the embryos by trypsinization. The cells were innoculated into carrel flasks and incubated in a tissue culture medium (TC-199) containing 10 % calf serum at 37.C. On the second or following days, secondary passages were made from the cells by innoculating them into T-15 culture flasks. The cells were incubated in tissue culture medium at 37.C for 48 hours. The medium was then decanted, the cells were washed with Balanced Salt Solution (B.S.S.), and tissue culture medium containing a specific amount of AET was added to the cells. The cells were incubated in this mixture for 30 minutes at 37.C. The medium was then withdrawn, the cells were washed 3 times with B.S.S., and new medium not containing AET was added to them.
Before the addition of AET and after its removal, the amount of cells in a specific area of the flask (0.16 mm2) was counted under the inverse microscope with the help ofa micrometric net and thus, the per cent of survival was calcula- ted.
In the experiments, the effect of AET for rat embryonic cells at concentrations of 16, 20, 25, 30, 32, 35, 40, 42, and 45 mM was investigated. For each con- centration, two sets of T-15 flasks were used and the above mentioned procedures were repeated. The control series were run in the same manner as the experimental ones and AET was not added to their medium.
Preparation of AET
The pH of the tissue culture medium was adjusted with 1 N NaOH (VOS
et ٠/., 1962) in such a way that after the addition of AET and serum the pH of
the final solution was 7.4. A specific amount of AET was then dissolved in this medium and 10 % calf scrum was added to this mixture. The medium was imme- diately transferred to T-15 flasks containing the cells.
147 TOXIC EFFECT OF AET
SERIE B, TOME XXXVII FASC. 3 - 4
RESULTS AND DISCUSSION
As will be seen in Table I, the per cent of surviving cells after the addition and removal of AET, is around 99.00 % at concentrations of 16, 20, and 25 mM. 15 minutes after the addition of 45 mM, almost 50 % and in 30 minutes none of the cells survived. At 25 mM 99.06% of the cells survived, but the condition of the cells, histologically, was better at 20 mM. For this reason, it was deter- mined that the highest non-toxic concentration of AET for rat embryonic cells was 20 mM and the lethal concentration was 45 mM.
Table I
The effect of AET (mM) on the survival of rat embryonic cells.
Concentration زﻻ Survival of of AET cells 16 mM 99.31 20 mM 99.00 25 mM 99.09 30 mM 96.16 32 mM ﺃ)1\1 35 mM 78.03 40 mM 66.71 42 mM 24.34 45 mM 0.00
In the forthcoming experiments, 20 mM of AET will be utilized to investi- gate its radioprotective effects against Co-60 irradiation and on tlie synthesis of nucleic acids and proteins of irradiated and unirradiated rat embryonic cells in tissue culture.
The authors would like to express their most sincere thanks to Prof. Dr. Atif ŞENGÜN and to Dr. Laszlo SZTANYIK (I.A.E.A.) for their helpful suggestions throughout the course of these experiments.
REFERENCES
BACQ, Z. M. and ALEXANDER, p. (1966) : Fundamentals o f Radiation Biology, (2 nd. ed.), 562 pp. New York : Pergamon Press.
DOHERTY, D .C ., BURNETT Jr., w . T., and SHAPIRA, R., (1957) : Mercaptoethylamines and related compounds with protective ability. Rad. Research, 7:13-21.
LEONARD, A. and MAISIN, j. R. (1964) : Effect of 2-d-aminoethylisothiourea (AET) against genetic damages induced by X-irradiation of male mice. Rad. Research, 23:52-62. MAISIN, J.R ., LEONARD, A. and HUGAN, j. (1965) : Tissue and cellular distribution of
tritium labelled AET in mice. j . Natl. Cancer Inst., 35: 103-109.
NEWSOME, j. R., KNOTT, D. H. and CVERMANN, R. R. (1962) : Radioprotective effects of/?-aminoethylisothiouronium Er. HBr in the dog. Red. Research, 17:847-354.
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NEWSOME, j. R. and OVERMAN, R. R. (1964) : The protective effects of (.'-aminocthyli- sothiorunium Br. HBr and p-aminopropiophenone against acute sublethal whole-body x-irradiation in the dogs. Rad. Research, 21:530-540.
PATT, H. M„ TYREE, E. B„ STRAOBE, R. I . and SMITH, D. E. (1949) : Cystein protection against X-irradiation. Science, 110:213-214.
VOS, O., BUDKE, 1. and VERGROESEN, A. j. (1962) : Protection of tissue culture against ionizing radiation. I. The effect of biological amines, disulphide compounds, and thiols.
Int. j . Rod, Biol.. 551-542.•؛ .
