Mutation Research, 31 (I975) IO3-IO8 © Elsevier Scientific Publishing Company, Amsterdam--Printed in The Netherlands
lO 3
T H E COMPARATIVE C Y T O G E N E T I C EFFECTS OF A L D R I N AND PHOSPHAMIDON
LILIANA GEORGIAN Victor Babes Institute of Pathology and Medical Genetics, Spl. Independentei 99-Io~ Bucharest (Rumania) (Received September 27th, 1974)
SUMMARY
An organochlorinated pesticide (aldrin) and an organophosphorus one (phosphamidon) were administered in human lymphocyte cultures, and the cytogenetic effects were related to the compound concentration. The comparative estimation of the number and type of chromosome aberrations observed in the treatments with various doses of drugs permits the following statements. (a) The aldrin showed a narrow range of clastogenic doses, between I9.125 and 38.25 /~g/ml. Since these doses are near the limit for cell survival, the observed chromosome lesions are probably not perpetuated in other abnormal cells. (b) Comparatively, the range of phosphamidon clastogenic doses is very large, scattered between 1. 9 and 122/~g/ml. Since, in the phosphamidon treatments, the cellular death begins at a concentration above 244 #g/ml, the chromosome aberrations, induced especially by the low doses, could be maintained in other abnormal cells. In a smaller number of experiments, chromosome examinations were performed after intraperitoneal injections of the drugs into rats and mice, 24 h before harvesting of the bone marrow. The administered doses were low, as compared with those of the experiments in vitro: the minimal doses inducing chromosome aberrations in vivo were, in the aldrin treatments 9.56, and in the phosphamidon treatments, o.o 7/~g/g body weight. In the experiments both in vitro and in vivo, the analysis of the frequencies of the abnormal cells and of the chromosome lesion types support the existence of a dose-response correlation. The genetic peril due to low doses of pesticides with a general weak toxic effect is discussed.
Since pesticides came into wide use, the possible genetic effects of such drugs has become of great interest. The known persistence of pesticide residues in the soil 1~,19 and in vegetable 15 and animal tissues1,7,1, represents a continuous poisoning source for man. There is also evidence for the persistence of some pesticide residues in human adipose tissues ~0. There is also peril due to occupational contact10,18, 21 or to acute intoxication s.
104
L. G E O R G I A N
Genetic studies on pesticide effects are concerned with their action on nucleic acidslL their effects on the mutagenic rate in host-mediated assays and with the dominant lethal method 5, the action on the chromosomes in vivo%~°,~%1%~8,21 and i n vit~'o %11,1%17,18 etc. This study deals with the action, in vitro and in vivo, of two pesticides: an organochlorinated compound, aldrin, and an organophosphorus one, phosphamidon, kindly supplied by Dr. E. Gabrielescu from the Hygiene Institute, Bucharest. As reference data in the selection of doses, we used the LDso values of the drugs as determined for rats. In the experiments with aldrin in vitro the following doses were administered: 76.5 ° (LDso value on the rat), 38.25 and 19.125/~g/ml. In the treatments in vivo doses for rats and mice were 76.5 o, 38.25, 19.125 and 9.56 #g/g body weight. In the treatments with phosphamidon in vitro the doses were 122, 61, 30.5 (LDso value on the rat), 15.25, 7.6, 3.8, and 1.8/~g/ml. For the treatments in vivo we used some very low doses because the doses administered in vitro killed the animals (rats and mice) in 2-5 h after their intraperitoneal application. In the Wistar rats, chromosome analyses were performed at the doses of 0.3, o.15 and o.075 #g/g body weight. In the A K R mice, the tolerated doses of phosphamidon were o.15 and 0.075 /~g/g body weight. These doses are very low compared with the LDs0 values for rats. As material for the experiments in vitro human peripheral blood cultures were used ~. The doses of the tested pesticides were administered for the last 22 h in each 72-h culture. Propyleneglycol (Carlo Erba) was used as organic solvent for aldrin and phosphamidon. In the control cultures an equivalent volume of solvent was added. Positive controls were similar 72-h cultures, treated for the last 22 h with mitomyein C (Kyowa) at i #g/ml. In the experiments in vivo, chromosome examinations were performed on the bone marrow cells of 5 rats and 5 miceS for each dose. The drugs were administered intraperitoneally 24 h before the harvesting of the bone marrow. As in the experiments in vitro, the pesticides were dissolved in propyleneglycol, and controls were injected with the solvent only. The cytogenetic abnormalities observed were scored and submitted to the statistical test of Z~. Under the term "chromosome abnormalities" in the 5th column of Table I, and in the 5th and 9th columns of Table II, both the chromosome and chromatid types of aberration are included. In the 9th and I l t h columns of Table I, chromosome types of aberration are referred to as gaps, breaks, deletions, fragments and interchanges involving the two chromatids, and to the chromatid types of aberrations as gaps, breaks, deletions, fragments and interchanges involving only one of the two chromatids. In the experiments in vitro, aldrin produced chromosome aberrations at the dose of 19.125 #g/ml. At the dose of 38.25 #g/ml, the cells died, and at the dose of 9.56/~g/ml no chromosome lesion was observed. The observed abnormalities were of both chromosome and chromatid type: gaps, breaks, deletions and fragments. Rearrangements were only rarely found. In treatments with phosphamidon in vitro a wide range of doses yielded chromosome aberrations. Moreover, it was possible to relate the effect on chromosomes to the compound concentration in the medium, and this correlation concerns not only
TABLE
I
39 123 22 119 155 77
84
273
273
Total number of observed cells
38 8o io 38 22 ii
16
77
3
in vitro
71 127 13 42 27 13
17
83
3
1.8o 1.o 3 0.59 0.35 o.i 7 o.16
0.20
0.3o
o.oi
Chromosome aberrations Observed P e r cell
IN THE EXPERIMENTS
Cells with chromosome aberrations observed
OF CHROMOSOME ABERRATIONS
76.5 ° 38.25
Aldrin
61 ioo 223
153
o.o7
18
i 22 33
19
i 22 35
21
o.12
o.oi 0.22 o.15
o.14
o.ii
63
2
E x p e r i m e n t s on mice Total Cells with number of chromosome observed aberrations cells observed
12
all a n i m a l s d e a d 15 I three animals dead 94 8
I2I
3° 8 three animals dead
3
0.02
O.I 5
I2
Z ~ = 48.16
0-07 0.74 0. 5 0.27 o.19
0.09
o.12
0.007
3 93 ii 33 17
8
43
26 3 three animals dead 145 20
i
Chromosome aberrations Observed P e r cell
48.7 ° 17.o 7 13.63 3.36 i .94 2.59
1.19
2.19
2
N u m b e r of chromatid types o f aberration Observed P e r cell
all a n i m a l s d e a d
I
rats Cells with chromosome aberrations observed
19 21 3 4 3 2
i
6
Cells with multiple chromosome aberrations ( M cell) Observed P e r cent
all a n i m a l s d e a d
5°
E x p e r i m e n t s on Total number of observed cells
9.56 0.30 o.15
19.125
--
Untreated
Phosphamidon
Dose in I~g/g body weight
Treatment
THE COMPARATIVE YIELD OF CHROMOSOME ABERRATIONS IN THE TREATMENTS in vivo
II
61 30.5 15.25 7.6 3.8 1.9
Phosphamidon
TABLE
19.125
Aldrin
I
--
Untreated
Mitomycin C
Dose (t~g/ml)
Treatment
THE COMPARATIVE YIELD
O.OI
-0.o8 0.09 o.07 o.o6
o.Io
o.II
o.o 3
IO
i
I2
io
2
o.io
o.07
0.O 9
o.3o
o.o 3
Chromosome aberrations Observed P e r cell
Z 2 - - 42.81
I
-io 2 9 io
9
4°
I
N u m b e r of chromosome types of aberration Observed P e r cell
H
o zJl
t~
oq
©
o~
t~
t~
I06
L. GEORGIAN
the number of abnormal cells but also the chromosome aberration types. At the high doses of 61 and also at 3o.5/zg/ml, a colchicine-like effect was observed, with short and thick chromatids and elongated centromeres, although the same doses administered 2 h before the cell harvesting produced no stathmokinetic effect. At the middle (15.25 and 7.6/~g/ml) and small (3.8 and 1. 9/,g/ml) doses, the observed types of aberration were chromatid and chromosome gaps, breaks, deletions and fragments. A number of chromosome and chromatid rearrangements were also observed. "Ihe comparative yield of chromatid and chromosome types of aberration presents a statistical significance expressed as )~2 (Table I). The comparative damaging action of the drugs on chromosomes was estimated as to their ability to induce M cells (cells with multiple chromosome aberrations). This analysis also showed significant differences between aldrin and the different doses of phosphamidon (Table I). The comparative estimation of the number and type of chromosome aberrations observed in the treatments with various doses of drugs permits the following statements. (a) The aldrin showed a narrow range of clastogenic doses, between 19.125 and 38.25/zg/rnl. Since these doses are near the limiting value lor cell survival, the observed chromosome lesions are probably not perpetuated in other abnormal cells. (b) Comparatively, the range of phosphamidon clastogenic doses is very large, scattered between 1. 9 and 122 #g/ml. Since, in the phosphamidon treatments, the cellular death begins at a concentration above 244/zg/ml, the chromosome aberrations, induced especially by the low doses, could be maintained in other abnormal cells. In a small number of experiments, chromosome examinations were performed after intraperitoneal injections of tile drugs into rats and mice, 24 h before the harvesting of bone marrow. The doses administered were low, as compared with those of the experiments in vitro: the minimal doses inducing chromosome aberrations i~, vivo were, in the aldrin treatments 9.56, and in the phosphamidon treatments, o.o 7/~g/g body weight. In the phosphamidon treatments, there was an evident difference in drug sensitivity between rats and mice. Whereas the highest tolerated dose in mice was o.o 7/~g/g body weight, in rats a 24-h survival was possible at o.3o/*g/g body weight (Table II). Possibly, the survival time could be much longer, but we did not study it. In the treatments with phosphamidon on rats in vivo all interchanges and most of the observed deletions were of the chromosome type, whereas most of the gaps, breaks and fragments were of the chromatid type. This could be due to the existence of some more closed interchromatid bridges, but this assumption requires further experiments. In the experiments with the two pesticides both in vivo and in vitro, there was a dose response relation concerning the number of aberrant cells and chromosome aberrations (Tables I and II). The types of induced chromosome abnormalities have also to be discussed because of their correlation with the administered concentrations of the drugs. In the phosphamidon treatments in vitro with 61 and 35.5 #g/ml a great number of C-like mitoses was observed. This m a y be due to an action on the spindle proteins, as similarly found after other treatments with pesticide 2& The dose-response effect is also expressed in terms of M-cell production, corresponding to high doses of phosphamidon
CYTOGENETIC EFFECTS OF PESTICIDES
107
(Table I). The existence of multiple chromosome abnormalities in the high dose treatm e n t s possibly reduces the chance of survival of the carrying cells. The presence of chromosome a n d c h r o m a t i d exchanges in the t r e a t m e n t s with low doses of p h o s p h a m i d o n i n vitro raises the question of the persistence of a b n o r m a l clones. Similar observations have been reported for other pesticide treatmentsg, TM. Because certain chromosome interchanges a n d small deletions are consistent with a n o r m a l cell division, t h e y m i g h t be t r a n s m i t t e d to the d a u g h t e r cells a n d thus produce a genetic hazard to somatic a n d possibly to gonadal cells. The observed a b i l i t y of the low doses of p h o s p h a m i d o n to produce chromosome aberrations i n vitro a n d especially the elastogenic action of very low doses i n vivo, have to be t a k e n into account. Also, the comparative cytogenetic effects of aldrin a n d p h o s p h a m i d o n treatm e n t s i n vitro raise the question of the possible genetic peril from pesticides with weak toxicity. The well tolerated low concentrations of some pesticides, for example phosphamidon, perhaps yield persistent chromosome abnormalities i n vivo. ACKNOWLEDGEMENTS I t h a n k Mrs. GRAPINA VRABIE a n d MARIANASUBA for their valuable technical assistance. I a m also grateful to Dr. ELIANA GABRIELESCU,from the Hygiene I n s t i t u t e , Bucharest, for k i n d l y supplying the pesticides.
REFERENCES I ADLEY,F. E., ANDW. O. GRANT,Mercury concentrations in game birds, State of Washington197° and 1971, Pesticide Monit. J., 6 (1972) 91-93. 2 AHMED,M., AND F. W. GRANT, Cytological effects of the mercurial fungicide, Panogen 15, on Tradescantia and Viciafaba root tips, Mutation Res., 14 (1972) 391-396. 3 ARAKAKI, O. T., AND S. R. SPARKERS, Microtechnique for culturing leukocytes from whole blood, Cytogenetics, 2 (1963) 57-6o. 4 BENSON, W. R., The chemistry of pesticides, Ann. N. Y. Acad. Sci., 16o (1969) 7-29. 5 BUSELMAIER, W., G. ROHRBORN AND P. PROPPING, Comparative investigations on the mutagenicity of isoniazid in mammalian test systems, (Abstract), Mutation Res., 21 (1973) 25-26. 6 CZEIZEL,A., TRINH VAN BAD, I. SZABO AND P. RUZICSKA, Human chromosome aberrations in acute organic phosphorous acid ester (pesticide) intoxication, (Abstract), Mutation Res., 21 (1973) I87-I88. 7 DUSTMAN, E. I-I., AND F. E. STICKEL,The occurrence and significance of pesticide residues in wild animals, Ann. N . Y . Acad. Sci., 16o (1969) I62-172. 8 FORD, E. H. R., AND M. H. D. WOOLLAM, A study on the mitotic chromosomes of mice of the strong A line, Exptl. Cell Res., 32 (1963) 320-326. 9 GRANT,W. F., Cytological effect of environmental mutagens--pesticides, (Abstract), Mutation Res., 21 (1973) 221-222. IO HOOPINGARNER, R., AND A. W. BLOOMER, Lymphocyte chromosome analysis of pesticide exposed individuals, Intern. Congr. Plant Protection, Paris, 7 (197o) 772. 1i HUANG,C. C., Effect on growth but not on chromosomes of the mammalian cells after treatment with three organophosphorus insecticides, Proc. Soc. Exptl. Biol. Med., 142 (1973) 36-4 o. 12 KELLY-GARVERT,F., and M. S. LEGATOR,Cytogenetic and mutagenic effects of DDT and DDE in a Chinese hamster cell line, Mutation Res., 17 (1973) 223-229. 13 LAWS, E. R., JR., A. CURLEY AND J. F. BIRDS, Men w i t h intense occupational exposure to
DDT. A clinical and chemical study, Arch. Environ. Health, 15 (1966) 766-775. 14 MOHN, G., Comprison of the mutagenic activity of eight organophosphorus insecticides in Escherichia colt, (Abstract), Mutation Res., 21 (1973) 196. 15 STEEVENS, D. R., M. L. WALSH AND R. ]). KENNEY, Arsenic residues in soil and potatoes from Wisconsin potato fields--i97o, Pesticide Monit. J., 6 (1972) 89-90. 16 STENERSEN, J., AND J. KVAKVAG, Residues of DDT and its degradation products in cod liver from two Norwegian fords, Bull. Environ. Contain. Toxicol., 8 (1972) 12o-121.
108
L. GEORGIAN
I 7 STYLES, J. A., C y t o t o x i c effects of v a r i o u s pesticides in vivo a n d in vitro, (Abstract), Mutation Res., 21 (1973) 5o-51 18 STYLES, J. A., M u t a g e n i c effects of b e n z i m i d a z o l e c a r b a m a t e (BCM) a n d related pesticides, (Abstract), M u t a t i o n Res., 21 (1973) 203. 19 ~¢VIERSMA, G. IS., H. TAI AND F. P. SAND, Pesticide residues in soil f r o m eight c i t i e s - - I 9 6 9 , Pesticide Monit. J . , 6 (1972) 126-129. 20 WYLLIE, J., J. GABICA AND W. W. BENSON, C o m p a r a t i v e o r g a n o c h l o r i n e pesticide residues in s e r u m a n d biopsied lipoid t i s s u e : a s u r v e y on 2oo p e r s o n s in s o u t h e r n I d a h o - 197 o, Pesticide, Monit. J., 6 (1972) 84-88. 21 YODER, J. M., M. WATSON AND W. W. BENSON, L y m p h o c y t e c h r o m o s o m e a n a l y s i s of agricult u r a l w o r k e r s d u r i n g e x t e n s i v e o c c u p a t i o n a l e x p o s u r e to pesticides, Mutation Res., 21 (1973) 335-34 ° .
lO 3
T H E COMPARATIVE C Y T O G E N E T I C EFFECTS OF A L D R I N AND PHOSPHAMIDON
LILIANA GEORGIAN Victor Babes Institute of Pathology and Medical Genetics, Spl. Independentei 99-Io~ Bucharest (Rumania) (Received September 27th, 1974)
SUMMARY
An organochlorinated pesticide (aldrin) and an organophosphorus one (phosphamidon) were administered in human lymphocyte cultures, and the cytogenetic effects were related to the compound concentration. The comparative estimation of the number and type of chromosome aberrations observed in the treatments with various doses of drugs permits the following statements. (a) The aldrin showed a narrow range of clastogenic doses, between I9.125 and 38.25 /~g/ml. Since these doses are near the limit for cell survival, the observed chromosome lesions are probably not perpetuated in other abnormal cells. (b) Comparatively, the range of phosphamidon clastogenic doses is very large, scattered between 1. 9 and 122/~g/ml. Since, in the phosphamidon treatments, the cellular death begins at a concentration above 244 #g/ml, the chromosome aberrations, induced especially by the low doses, could be maintained in other abnormal cells. In a smaller number of experiments, chromosome examinations were performed after intraperitoneal injections of the drugs into rats and mice, 24 h before harvesting of the bone marrow. The administered doses were low, as compared with those of the experiments in vitro: the minimal doses inducing chromosome aberrations in vivo were, in the aldrin treatments 9.56, and in the phosphamidon treatments, o.o 7/~g/g body weight. In the experiments both in vitro and in vivo, the analysis of the frequencies of the abnormal cells and of the chromosome lesion types support the existence of a dose-response correlation. The genetic peril due to low doses of pesticides with a general weak toxic effect is discussed.
Since pesticides came into wide use, the possible genetic effects of such drugs has become of great interest. The known persistence of pesticide residues in the soil 1~,19 and in vegetable 15 and animal tissues1,7,1, represents a continuous poisoning source for man. There is also evidence for the persistence of some pesticide residues in human adipose tissues ~0. There is also peril due to occupational contact10,18, 21 or to acute intoxication s.
104
L. G E O R G I A N
Genetic studies on pesticide effects are concerned with their action on nucleic acidslL their effects on the mutagenic rate in host-mediated assays and with the dominant lethal method 5, the action on the chromosomes in vivo%~°,~%1%~8,21 and i n vit~'o %11,1%17,18 etc. This study deals with the action, in vitro and in vivo, of two pesticides: an organochlorinated compound, aldrin, and an organophosphorus one, phosphamidon, kindly supplied by Dr. E. Gabrielescu from the Hygiene Institute, Bucharest. As reference data in the selection of doses, we used the LDso values of the drugs as determined for rats. In the experiments with aldrin in vitro the following doses were administered: 76.5 ° (LDso value on the rat), 38.25 and 19.125/~g/ml. In the treatments in vivo doses for rats and mice were 76.5 o, 38.25, 19.125 and 9.56 #g/g body weight. In the treatments with phosphamidon in vitro the doses were 122, 61, 30.5 (LDso value on the rat), 15.25, 7.6, 3.8, and 1.8/~g/ml. For the treatments in vivo we used some very low doses because the doses administered in vitro killed the animals (rats and mice) in 2-5 h after their intraperitoneal application. In the Wistar rats, chromosome analyses were performed at the doses of 0.3, o.15 and o.075 #g/g body weight. In the A K R mice, the tolerated doses of phosphamidon were o.15 and 0.075 /~g/g body weight. These doses are very low compared with the LDs0 values for rats. As material for the experiments in vitro human peripheral blood cultures were used ~. The doses of the tested pesticides were administered for the last 22 h in each 72-h culture. Propyleneglycol (Carlo Erba) was used as organic solvent for aldrin and phosphamidon. In the control cultures an equivalent volume of solvent was added. Positive controls were similar 72-h cultures, treated for the last 22 h with mitomyein C (Kyowa) at i #g/ml. In the experiments in vivo, chromosome examinations were performed on the bone marrow cells of 5 rats and 5 miceS for each dose. The drugs were administered intraperitoneally 24 h before the harvesting of the bone marrow. As in the experiments in vitro, the pesticides were dissolved in propyleneglycol, and controls were injected with the solvent only. The cytogenetic abnormalities observed were scored and submitted to the statistical test of Z~. Under the term "chromosome abnormalities" in the 5th column of Table I, and in the 5th and 9th columns of Table II, both the chromosome and chromatid types of aberration are included. In the 9th and I l t h columns of Table I, chromosome types of aberration are referred to as gaps, breaks, deletions, fragments and interchanges involving the two chromatids, and to the chromatid types of aberrations as gaps, breaks, deletions, fragments and interchanges involving only one of the two chromatids. In the experiments in vitro, aldrin produced chromosome aberrations at the dose of 19.125 #g/ml. At the dose of 38.25 #g/ml, the cells died, and at the dose of 9.56/~g/ml no chromosome lesion was observed. The observed abnormalities were of both chromosome and chromatid type: gaps, breaks, deletions and fragments. Rearrangements were only rarely found. In treatments with phosphamidon in vitro a wide range of doses yielded chromosome aberrations. Moreover, it was possible to relate the effect on chromosomes to the compound concentration in the medium, and this correlation concerns not only
TABLE
I
39 123 22 119 155 77
84
273
273
Total number of observed cells
38 8o io 38 22 ii
16
77
3
in vitro
71 127 13 42 27 13
17
83
3
1.8o 1.o 3 0.59 0.35 o.i 7 o.16
0.20
0.3o
o.oi
Chromosome aberrations Observed P e r cell
IN THE EXPERIMENTS
Cells with chromosome aberrations observed
OF CHROMOSOME ABERRATIONS
76.5 ° 38.25
Aldrin
61 ioo 223
153
o.o7
18
i 22 33
19
i 22 35
21
o.12
o.oi 0.22 o.15
o.14
o.ii
63
2
E x p e r i m e n t s on mice Total Cells with number of chromosome observed aberrations cells observed
12
all a n i m a l s d e a d 15 I three animals dead 94 8
I2I
3° 8 three animals dead
3
0.02
O.I 5
I2
Z ~ = 48.16
0-07 0.74 0. 5 0.27 o.19
0.09
o.12
0.007
3 93 ii 33 17
8
43
26 3 three animals dead 145 20
i
Chromosome aberrations Observed P e r cell
48.7 ° 17.o 7 13.63 3.36 i .94 2.59
1.19
2.19
2
N u m b e r of chromatid types o f aberration Observed P e r cell
all a n i m a l s d e a d
I
rats Cells with chromosome aberrations observed
19 21 3 4 3 2
i
6
Cells with multiple chromosome aberrations ( M cell) Observed P e r cent
all a n i m a l s d e a d
5°
E x p e r i m e n t s on Total number of observed cells
9.56 0.30 o.15
19.125
--
Untreated
Phosphamidon
Dose in I~g/g body weight
Treatment
THE COMPARATIVE YIELD OF CHROMOSOME ABERRATIONS IN THE TREATMENTS in vivo
II
61 30.5 15.25 7.6 3.8 1.9
Phosphamidon
TABLE
19.125
Aldrin
I
--
Untreated
Mitomycin C
Dose (t~g/ml)
Treatment
THE COMPARATIVE YIELD
O.OI
-0.o8 0.09 o.07 o.o6
o.Io
o.II
o.o 3
IO
i
I2
io
2
o.io
o.07
0.O 9
o.3o
o.o 3
Chromosome aberrations Observed P e r cell
Z 2 - - 42.81
I
-io 2 9 io
9
4°
I
N u m b e r of chromosome types of aberration Observed P e r cell
H
o zJl
t~
oq
©
o~
t~
t~
I06
L. GEORGIAN
the number of abnormal cells but also the chromosome aberration types. At the high doses of 61 and also at 3o.5/zg/ml, a colchicine-like effect was observed, with short and thick chromatids and elongated centromeres, although the same doses administered 2 h before the cell harvesting produced no stathmokinetic effect. At the middle (15.25 and 7.6/~g/ml) and small (3.8 and 1. 9/,g/ml) doses, the observed types of aberration were chromatid and chromosome gaps, breaks, deletions and fragments. A number of chromosome and chromatid rearrangements were also observed. "Ihe comparative yield of chromatid and chromosome types of aberration presents a statistical significance expressed as )~2 (Table I). The comparative damaging action of the drugs on chromosomes was estimated as to their ability to induce M cells (cells with multiple chromosome aberrations). This analysis also showed significant differences between aldrin and the different doses of phosphamidon (Table I). The comparative estimation of the number and type of chromosome aberrations observed in the treatments with various doses of drugs permits the following statements. (a) The aldrin showed a narrow range of clastogenic doses, between 19.125 and 38.25/zg/rnl. Since these doses are near the limiting value lor cell survival, the observed chromosome lesions are probably not perpetuated in other abnormal cells. (b) Comparatively, the range of phosphamidon clastogenic doses is very large, scattered between 1. 9 and 122 #g/ml. Since, in the phosphamidon treatments, the cellular death begins at a concentration above 244/zg/ml, the chromosome aberrations, induced especially by the low doses, could be maintained in other abnormal cells. In a small number of experiments, chromosome examinations were performed after intraperitoneal injections of tile drugs into rats and mice, 24 h before the harvesting of bone marrow. The doses administered were low, as compared with those of the experiments in vitro: the minimal doses inducing chromosome aberrations i~, vivo were, in the aldrin treatments 9.56, and in the phosphamidon treatments, o.o 7/~g/g body weight. In the phosphamidon treatments, there was an evident difference in drug sensitivity between rats and mice. Whereas the highest tolerated dose in mice was o.o 7/~g/g body weight, in rats a 24-h survival was possible at o.3o/*g/g body weight (Table II). Possibly, the survival time could be much longer, but we did not study it. In the treatments with phosphamidon on rats in vivo all interchanges and most of the observed deletions were of the chromosome type, whereas most of the gaps, breaks and fragments were of the chromatid type. This could be due to the existence of some more closed interchromatid bridges, but this assumption requires further experiments. In the experiments with the two pesticides both in vivo and in vitro, there was a dose response relation concerning the number of aberrant cells and chromosome aberrations (Tables I and II). The types of induced chromosome abnormalities have also to be discussed because of their correlation with the administered concentrations of the drugs. In the phosphamidon treatments in vitro with 61 and 35.5 #g/ml a great number of C-like mitoses was observed. This m a y be due to an action on the spindle proteins, as similarly found after other treatments with pesticide 2& The dose-response effect is also expressed in terms of M-cell production, corresponding to high doses of phosphamidon
CYTOGENETIC EFFECTS OF PESTICIDES
107
(Table I). The existence of multiple chromosome abnormalities in the high dose treatm e n t s possibly reduces the chance of survival of the carrying cells. The presence of chromosome a n d c h r o m a t i d exchanges in the t r e a t m e n t s with low doses of p h o s p h a m i d o n i n vitro raises the question of the persistence of a b n o r m a l clones. Similar observations have been reported for other pesticide treatmentsg, TM. Because certain chromosome interchanges a n d small deletions are consistent with a n o r m a l cell division, t h e y m i g h t be t r a n s m i t t e d to the d a u g h t e r cells a n d thus produce a genetic hazard to somatic a n d possibly to gonadal cells. The observed a b i l i t y of the low doses of p h o s p h a m i d o n to produce chromosome aberrations i n vitro a n d especially the elastogenic action of very low doses i n vivo, have to be t a k e n into account. Also, the comparative cytogenetic effects of aldrin a n d p h o s p h a m i d o n treatm e n t s i n vitro raise the question of the possible genetic peril from pesticides with weak toxicity. The well tolerated low concentrations of some pesticides, for example phosphamidon, perhaps yield persistent chromosome abnormalities i n vivo. ACKNOWLEDGEMENTS I t h a n k Mrs. GRAPINA VRABIE a n d MARIANASUBA for their valuable technical assistance. I a m also grateful to Dr. ELIANA GABRIELESCU,from the Hygiene I n s t i t u t e , Bucharest, for k i n d l y supplying the pesticides.
REFERENCES I ADLEY,F. E., ANDW. O. GRANT,Mercury concentrations in game birds, State of Washington197° and 1971, Pesticide Monit. J., 6 (1972) 91-93. 2 AHMED,M., AND F. W. GRANT, Cytological effects of the mercurial fungicide, Panogen 15, on Tradescantia and Viciafaba root tips, Mutation Res., 14 (1972) 391-396. 3 ARAKAKI, O. T., AND S. R. SPARKERS, Microtechnique for culturing leukocytes from whole blood, Cytogenetics, 2 (1963) 57-6o. 4 BENSON, W. R., The chemistry of pesticides, Ann. N. Y. Acad. Sci., 16o (1969) 7-29. 5 BUSELMAIER, W., G. ROHRBORN AND P. PROPPING, Comparative investigations on the mutagenicity of isoniazid in mammalian test systems, (Abstract), Mutation Res., 21 (1973) 25-26. 6 CZEIZEL,A., TRINH VAN BAD, I. SZABO AND P. RUZICSKA, Human chromosome aberrations in acute organic phosphorous acid ester (pesticide) intoxication, (Abstract), Mutation Res., 21 (1973) I87-I88. 7 DUSTMAN, E. I-I., AND F. E. STICKEL,The occurrence and significance of pesticide residues in wild animals, Ann. N . Y . Acad. Sci., 16o (1969) I62-172. 8 FORD, E. H. R., AND M. H. D. WOOLLAM, A study on the mitotic chromosomes of mice of the strong A line, Exptl. Cell Res., 32 (1963) 320-326. 9 GRANT,W. F., Cytological effect of environmental mutagens--pesticides, (Abstract), Mutation Res., 21 (1973) 221-222. IO HOOPINGARNER, R., AND A. W. BLOOMER, Lymphocyte chromosome analysis of pesticide exposed individuals, Intern. Congr. Plant Protection, Paris, 7 (197o) 772. 1i HUANG,C. C., Effect on growth but not on chromosomes of the mammalian cells after treatment with three organophosphorus insecticides, Proc. Soc. Exptl. Biol. Med., 142 (1973) 36-4 o. 12 KELLY-GARVERT,F., and M. S. LEGATOR,Cytogenetic and mutagenic effects of DDT and DDE in a Chinese hamster cell line, Mutation Res., 17 (1973) 223-229. 13 LAWS, E. R., JR., A. CURLEY AND J. F. BIRDS, Men w i t h intense occupational exposure to
DDT. A clinical and chemical study, Arch. Environ. Health, 15 (1966) 766-775. 14 MOHN, G., Comprison of the mutagenic activity of eight organophosphorus insecticides in Escherichia colt, (Abstract), Mutation Res., 21 (1973) 196. 15 STEEVENS, D. R., M. L. WALSH AND R. ]). KENNEY, Arsenic residues in soil and potatoes from Wisconsin potato fields--i97o, Pesticide Monit. J., 6 (1972) 89-90. 16 STENERSEN, J., AND J. KVAKVAG, Residues of DDT and its degradation products in cod liver from two Norwegian fords, Bull. Environ. Contain. Toxicol., 8 (1972) 12o-121.
108
L. GEORGIAN
I 7 STYLES, J. A., C y t o t o x i c effects of v a r i o u s pesticides in vivo a n d in vitro, (Abstract), Mutation Res., 21 (1973) 5o-51 18 STYLES, J. A., M u t a g e n i c effects of b e n z i m i d a z o l e c a r b a m a t e (BCM) a n d related pesticides, (Abstract), M u t a t i o n Res., 21 (1973) 203. 19 ~¢VIERSMA, G. IS., H. TAI AND F. P. SAND, Pesticide residues in soil f r o m eight c i t i e s - - I 9 6 9 , Pesticide Monit. J . , 6 (1972) 126-129. 20 WYLLIE, J., J. GABICA AND W. W. BENSON, C o m p a r a t i v e o r g a n o c h l o r i n e pesticide residues in s e r u m a n d biopsied lipoid t i s s u e : a s u r v e y on 2oo p e r s o n s in s o u t h e r n I d a h o - 197 o, Pesticide, Monit. J., 6 (1972) 84-88. 21 YODER, J. M., M. WATSON AND W. W. BENSON, L y m p h o c y t e c h r o m o s o m e a n a l y s i s of agricult u r a l w o r k e r s d u r i n g e x t e n s i v e o c c u p a t i o n a l e x p o s u r e to pesticides, Mutation Res., 21 (1973) 335-34 ° .
