Mutation Research, 40 (1976) 191--196 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
MUTAGENICITY AND TOXICITY'OF AMITROLE. II. HUMAN LYMPHOCYTE CULTURE TESTS
TYTTI MERETOJA, ULLA GRIPENBERG, DENNISBAMFORD, IRJA LAAMANENand MARJA SORSA Department of Genetics, University of Helsinki, SF-O0100 Helsinki 10 (Finland)
(Received September 12th, 1975) (Reveision ReceivedFebruary 9th, 1976) (Accepted March 1st, 1976)
Summary Effects of amitrole (3-amino-l,2,4-triazole) on human leucocytes in culture were investigated. Amitrole interfered with lymphoblast transformation and inhibited cell growth in concentrations of 0.2% w/v and higher. Selected metaphases were examined for th e presence of chromosome and chromatid aberrations. No clastogenic effects were observed.
Introduction The toxicity of amitrole to mammals appears to be very low. In rats the LDs0 value is 14.7 g/kg . Examination of fertility in treated animals revealed no demonstrable effects of amitrole . Neither was the incidence of congenital malformations affected. Long-term feeding experiments, however, resulted in the development of adenoma or adenocarcinoma of the thyroid gland . During these experiments high doses of amitrole were given during 24 months without interruption. Information about the effects of amitro!e on man is limited to one case where a person in a suicide attempt had taken a dose of 20 mg/kg . No toxic effects were foimd a n d amitrole was shown to be rapidly excreted in the urine. Only little is known about the effects of amitrole on human cells and tissues. Experimental work with amitrole on mammalian cells in vitro has, t o our knowledge, n o t y e t been performed. The purpose of this work was to study t h e genetically and cytologically damaging effects in conditions in vitro as part of a wider test programme, with the aim to decipher the mutagenic properties of this herbicide, which is currently in wide use:Short-term human blood cultures were chosen for testing amitrole on mammalian cells. Attention was paid to the
192 effect of amitrole on lymphoblast transformation in the blood cultures as well as on alterations in the morphological structure of the chromosomes. Material and m e t h o d s Whole blood microcultures were established according to conventional methods. To minimize variation in the quality of the preparations, blood from the same healthy female donor was used through all the experiments. An incubation time of 72 h was chosen to allow a maximal n u m b e r of the stimulated, although possibly inhibited, cells to reach the mitotic stage. The air-dried slides were stained with a 10% solution of Giemsa stain. In the first set of experiments the amitrole powder (purity 93.2--96.3%), dissolved in water, was added to the vessels during the whole culture time. A series of concentrations from 0.00001% to 1.0% w/v was used. In a second series of cultures, amitrole in high concentrations was added to the cell cultures only for the last 24, 6 and 2 h of growth. For each concentration 1--3 cultures were set up, each consisting of several vessels. Corresponding volumes of water were given to parallel control cultures. The mitotic index (the number of mitoses per 10000 cells) was counted for test and control cultures to measure the c y t o t o x i c i t y of amitrole. Metaphase plates were selected for detailed chromosome analysis. The number of aneuploid metaphases and the occurrence of structural chromosome and chromatid aberrations, including breaks and fragments, were carefully recorded. Results
Cytotoxic effects of amitrole The effects of various concentrations of amitrole t h a t affected the cells during the whole culture time was tested. Fig. 1 shows the dependence of the mitotic index on the different concentrations of amitrole. The concentration of 0.2% w/v turned out to be critical. At this concentration, lymphoblast transformation seemed to be inhibited. The mitotic index was lowered to only a third of the expected value. Lower concentrations of amitrole had no significant effect on the mitotic index. When higher concentrations were used, the mitotic index was clearly going down and the number of blast-like cells in the preparations seemed to decline. In addition, an increasing a m o u n t of cell debris was observed on the slides. In the second set of experiments, amitrole was added to the cultures only for the last hours of growth. As Fig. 2 shows, the cells were then able to stand much higher concentrations of amitrole. The toxic concentration of 0.2% w/v had no effect on the mitotic index when added to the culture for the last 24, 6 or2h.
Clastogenic effects of amitrole Amitrole in a concentration of 1% w/v is a highly c y t o t o x i c dose for human cells in culture. However, when added to the cell suspensions only for the last 24 and 6 h of growth, respectively, lymphoblast transformation is most probably already terminated. Although the toxic effect is considerable at this con-
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AMITROLE CONCENTRATION % (W/V) Fig. 1. Dependence of the mitotic index ratio (the ratio of mitotic indexes in amltrole and control cultures) on the concentration of amitrole added to the culture for the whole per/od of growth.
centration, a reduced number of metaphase plates could still be found to allow detailed chromosome analyses. As Table I shows, no significant difference between test cultures and controls was observed with respect to the occurrence of aneuploidy and structural changes of the chromosomes. This is true for the t w o different concentrations of amitrole (0.2 and 1.0% w/v) affecting the cells during the last hours of growth as well as during the whole culture time. Discussion Amitrole seems to inhibit lymphoblast transformation in blood cultures in concentration of 0.2% w/v and higher. However, the chromosome number as
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Fig. 2. The sign/flcance of time to the mitotic inhibition effect of amitrole. Mitotic index ratio is giving the ratio of mitotic indexes in amitrole and control cultures.
A n e u p l o i d cells
Concn % (w/v)
Cells w i t h c h r o m o s o m a l aberratinns
b r e a k , gap
gap, i s o c h r o -
gap, 2 e h r o m a t i d breaks
T y p e of a b e r r a t i o n
THE O C C U R R E N C E OF A N E U P L O I D Y AND C H R O M O S O M A L A B E R R A T I O N S IN M E T A P H A S E CELLS FROM C U L T U R E S C O N T A I N I N G TOXIC CONCENTRATIONS OF AMITROLE
well as the chromosome structure in the dividing cells seems to be totally unaffected. An explanation of the basic causes for the effects of amitrole on the blast cell transformation requires further investigation. The use of labelled aminotriazole would disclose the capacity of the agent to penetrate the cell membrane. Several reasons for the effect observed may be suggested. It is possible that amitrole is able to cause: (1) inhibition of enzymes needed for lymphoblast transformation. (2) deprivation of metabolites necessary for cells in culture, and (3) inactivation of phytohaemagglutinin causing the transformation process. The effect may also be due to impurities such as heavy metal ions in the amitrole sample used. Damage to the chromosomal structure indicates underlying changes of the genetic structure. According to our experiments, amitrole does not cause breakage of the chromosomes. However, the absence of visible chromosome damage does prove not the innocuity of amitrole with regard to mutagenicity and carcinogenicity. On the other hand, the results presented here give no support to the suspected chromosome damaging effects of this agent.
Acknowledgements Amitrole was provided by the State Institute of Agricultural Chemistry, Finland. This project has been supported by research grants from the National Research Council for Sciences (Academy of Finland). References 1 Geldmacher-Von MaUinekrodt, M. and H.P. Schmidt, Zur ToxicitAt und Stoffwechsel von A m i n o t r i a z o l beim Menschen, Arch. Toxikol., 27 (1970) 13--18. 2 Hapke, H.-J., Die Giftigkeit yon Aminotriazol filr Haustiere, Zbl. Veterinaermed., 14 (1967) 469--486. 3 Jukes, T.H. and C.B. Shaffer, Antithyroid effects of aminotrlazole, Science, 132 (1960) 296--297. 4 The Merck Index, Eighth Edition, Merck and Co., Inc. Rahway, N.J., U.S.A., 1968, p. 63.