107
Mutation Research, 40 (1976) 107--118 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
T H E E F F E C T OF INSECTICIDES ON CHINESE HAMSTER CELL C U L T U R E S
ULRIKE MAHR and HERBERT GEORG MILTENBURGER Institu te of Zoology, Technische Hochschule Darmstadt, D-6100 Darmstadt (Germany)
(Received May 21st, 1975) (Revision received November 18th, 1975) (Accepted November 28th, 1975) Summary The effect o f p,p'-isomers of DDT and its derivatives DDD, DDE and DDA on Chinese hamster cells in culture was studied. At different concentrations and various times o f t r e a t m e n t the proliferation rate was inhibited most strongly by DDD and DDT, whereas DDE exhibited a markedly weaker influence. DDA was the least toxic c o m p o u n d of the four. The cytogenetic effects were also different. Again, DDA induced the least damage. Only enhanced gap rates b u t no c h r o m o s o m e breaks were observed. DDE Was more active, and higher break rates occurred. DDD and DDT were by far the m ost damaging compounds, and t h e y raised the gap and break rates markedly. However, no induction of configuration anomalies was f o u n d in any experiment. Chronic t r e a t m e n t of the cells for 3 m o n t h s with DDT at 8 ppm did n o t alter the proliferation rate, the sensitivity to acute t r e a t m e n t with higher DDT concentrations or th e c h r o m o s o m a l aberration rates. The results are discussed in relation to the relevance of differential pesticide effectivity in organs of higher animals and man.
Introduction During the last 20 years a world-wide accumulation of pesticides in the organs o f m a n y species has been observed. Analyses with different test organisms showed th at DDT (dichloro-diphenyl-trichloroethane) and its metabolic derivatives can induce cellular damage. Though short c o n t a c t at concentrations normally present in the e n v i r o n m e n t did n o t p r o d u c e a measurable effect in man, chronic uptake proved t o be toxic [ 1 0 , 1 8 , 2 0 , 2 8 ] . Under long term exposure conditions the half-value times for the elimination of the pesticides are rather long, sometimes 4 years and more [ 1 1 ] . Most of the water-insoluble pesticides exist as micro-suspensions or form aerosols in the atmosphere. In the latter
108 form they fall to the earth with rain, consequently appearing in the terrestrial water circulation and later in food chains [7,51]. At present the uptake of pesticides is still greater than their elimination in most organisms. In cultures of kangaroo rat cells [44] and in other cell systems in vitro a toxic effect has been demonstrated. Gabliks and Friedman [17] found that 50% of HeLa and Chang liver cells die after a 48-h treatment with DDT (100 ppm). Johnson and Weiss [25] described a 50% proliferation inhibition in HeLa- and KB-cell cultures with a nutrient medium containing 25 pg DDA. Chung et al. [5,6] described a decrease in the cell number and an increase in the protein synthesis in HeLa cells with as little as 0.5 ppm DDT. A short treatment of 4.5 h with DDT (125 ppm) influenced the DNA and RNA synthesis of HeLa cells markedly [34], chicken cells became necrotic, and mitotic divisions were inhibited [47]. In another report, chicken cell cultures did not show signs of damage after treatment with DDT at 600 ppm [32]. The accumulation of metabolic derivatives of DDT in the tissues of higher organisms and the continued use of high levels of the insecticide as a consequence of increased levels of resistance had led to concern not only about the toxicological aspects but also about the possibility of mutagenic action by the metabolites [2,3,13,39,43]. The toxicological, physiological and genetical effects of DDT derivates DDD (dichloro-diphenyl-dichloroethane), DDE (dichloro-diphenyl-dichloroethene) and DDA (dichloro-diphenyl-acetate) have been examined in warm-blooded animals as well as in microorganisms [2,8,19,24,45,46]. The kind of damage varies markedly. Cytogenetic investigations on the effect of pesticides have produced inconsistent results. Experiments on Drosophila did not show mutagenicity of DDT [35]. Investigations using p,p' isomers of DDT, DDD, DDE, DDA and DDOM (2,2-bis(p,chlorophenyl)-ethanol(1)), another metabolite of DDT, resulted in a significant mutagenic effect only for DDA and a weak effect for DDT. The other compounds showed no effect [49]. Comparison of the action of p,p' and o,p' isomers of DDT and the metabolites DDD, DDE and DDA demonstrated a higher effectiveness of thep,p' isomers, which produced twice as many chromosomal aberrations as the o,p' isomers. DDT, DDD, DDE and DDA again produced different effects in the dominantlethal test and in the host-mediated assay. With the latter m e t h o d only DDD proved to be mutagenic, whereas none of the four compounds had any effect in the dominant4ethal test [4]. These results underline the c o m m o n experience in so far as the results may differ depending on the test system and the methods applied. Therefore, we felt it would be worth while to compare the way in which the proliferation-inhibiting and cytogenetic effects of DDT, DDD, DDE and DDA apply to mammalian cells in a standardizable system in vitro. We used a quasi-diploid cell line of the Chinese hamster, having 22/23 chromosomes in the stem line. Materials and methods
Cell cultures Cells of the Chinese hamster cell line B14 F28 were cultivated in 15 ml of
109 McCoy's medium [38] prepared in our laboratory, supplemented with 10% calf serum (GIBCO, New York) and Neomycin sulfate (Byk-Gulden, Konstanz) at 100 mg/1. The culture vessels were 180 ml glass bottles. The trypsinized (0.075% trypsin) cells were transferred to T-30 plastic flasks (FALCON, Los Angeles), and each flask was inoculated with 1200--1500 cells in 5 ml medium. After 4 h the cells had sedimented to the b o t t o m of the flask, where they adhered firmly. The flasks were then completely filled with medium and inverted. With a writing diamond, 50 to 100 single cells were encircled on the outer surface of a flask. Thus, within a circle each cell and its progeny could be observed during the following days, and individual colony growth curves could be established. The hanging position of the cells permitted the use of a normal microscope. Also, dead cells, losing contact with the vessel surface, fell to the opposite side (the b o t t o m ) of the flask, thereby disappearing from the microscopic field. Healthy and non-lethally damaged cells in interphase as well as in mitosis adhered firmly to the inner flask surface while "hanging". Comparative studies did n o t reveal any difference in cell multiplication or colony proliferation for "hanging" and "sitting" cultures. Another advantage of our m e t h o d is that only normally attached cells were analyzed. The plating efficiency was therefore consistent from experiment to experiment.
Pesticide treatment The lipophilic compounds DDT (Roth, Karlsruhe), DDD and DDE (Riedel De Haen, Hannover), not soluble in water, are easily dissolved in dimethylsulfoxide (DMSO; Merck, Darmstadt), which in turn is suspendable in water. In a series of experiments it was confirmed that the final concentration of 1% DMSO in the medium had no influence on the pesticide action. The pesticides were used as p,p' isomers mostly in the following concentrations: DDT 12, 24, 49 and 81 ppm; DDD 11, 22, 45 and 75 ppm; DDE 11, 22, 44 and 88 ppm; DDA 20, 41, 50, 68 and 100 ppm. DDA (Riedel De Haen, Hannover), which is water soluble, was also dissolved in DMSO for better comparison -- as to effects -- with the other substances. Owing to formation of many granules and vacuoles in cells after more than 4 h of treatment with the higher concentrations of the 4 substances, one series of experiments was run for only 4 h. When more than 90% of the cells had adhered to the vessel surface and spread out, the treatment was started, so that •the cells, but not their progeny, were treated with the pesticide. The pesticide medium was replaced by normal medium directly after the treatment time. In another series of experiments the pesticide was present in the medium for 4 days, so that the cells and their progeny were treated over the whole experimental time. Only m e d i u m concentrations in the range of 40--50 ppm were used. In a chronic treatment, the medium of the stock cultures continuously contained DDT at 8 ppm for a period of 3 months, a dose that occurs in mammals and man by accumulation over m a n y years [20,26,29,30]. These cells were then treated with DDT at 49 ppm for 4 days to test whether changes in resistance or sensitivity would occur.
Evaluation of effects The colony sizes, determined by counting the number of cells each day, were
110 grouped into classes of the theoretically possible cell numbers per generation: 1st generation, 2 cells; 2nd generation, 4 cells; 3rd generation, 8 cells etc. From this colony size, distribution curves were established referring to individual colonies and to the mean cell number out of all colonies of an experiment. The cell counts were done daily from day 0 (day of plating) to day 4, and the colonies were classified as those with 1, 2, 3--4, 5--8, 9--16 cells etc. Each value in the distribution curves represents the percentage out of at least 100 colonies. For the evaluation of the cytogenetic effect the metaphase cells were accumulated by 0.068 pg colcemid per ml medium (CIBA, Basel) given 4 h before chromosome preparation, followed by the normal air-drying procedure. Per dose and time at least 100 metaphases were evaluated on coded slides for structural aberrations: chromatid gaps (unstained area in a chromatid being smaller than a chromatid diameter or equal to it), chromatid breaks (opening in the chromatid being greater than the diameter or the chromatid piece was dislocated), including terminal deletions. As none of the four compounds induced increased aberration rates for rings, exchanges, dicentrics etc., such data are omitted from the following description. From the number of chromosomes in all cells scored having 19--25 chromosomes, mean chromosome distribution curves were established for evaluating possible variations of the numerical aberration rates. The cells were treated with different pesticide concentrations and for various exposure times (Tables II and III). For better comparison of an eventual c o m p o u n d action at exposure times of more than 4 h, nearly equal concentrations were tested. The ×2 test was used for statistical analysis. All data were regarded as different from the control when P ~ 0.01. Results
Colony damage The 4-h treatment revealed a differential effect of the four compounds on proliferation characteristics of the colonies during the 4 days after the start of the experiment. DDD-treated cells were damaged most. The effect could still be seen clearly on the 4th day, especially when the highest concentration of 75 ppm was used, even though the medium with the pesticide was replaced by normal medium directly after the 4-h treatment (Fig. 1A). The distribution curve of colony sizes was therefore shifted towards smaller cell numbers, and a m a x i m u m was n o t clearly formed (Fig. 1B). A b o u t 65% of the colonies consisted of not more than 8 cells, and 25% of the cells were lethally damaged as measured by counting the cell-free circles engraved on the outer surface of the flasks. A dose-dependent inhibitory effect on colony growth was also induced with the 45 and 22 ppm concentrations of DDD, but 11 ppm was ineffective. The high doses of DDT (81 ppm) and DDE (88 ppm) influenced the colony growth rate as well but n o t as much, and DDA (100 ppm) did not produce any effect. The medium and low concentrations of these compounds had only weak or no effect. Fig. 2 shows the results of the 4-day treatment presented as colony distribution curves. Again DDD was most effective, inducing a heavy proliferation inhibition. DDD (75 ~pm) killed 96% of the cells and no colony consisted of more than 4
111
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Fig. 2. C o l o n y size d i s t r i b u t i o n c u r v e s ; 4 - d a y t r e a t m e n t . A b s c i s s a : cells p e r c o l o n y in l o g a r i t h m i c scale. (o o) c o n t r o l ; (o I ) p p m D D T 1 2 ; D D D 1 1 ; D D E 1 1 ; D D A 2 0 ; (4 4) p p m D D T 2 4 ; D D D 2 2 ; D D E 2 2 ; D D A 4 1 ; (m m) p p m D D T 4 9 ; D D D 4 5 ; D D E 4 4 ; D D A 5 0 ; (0 ,) ppm DDT 8 1 ; D D D 7 5 ; D D E 8 8 ; D D A 6 8 ; ([] D) p p m D D A 1 0 0 .
112
cells. This ef f ect is d e m o n s t r a t e d by the colony size distribution curves where the maxima are drastically shifted towards the smaller cell numbers (Fig. 2B). Even after one day, DDD (75 ppm) killed more than 40% of the cells, which then disappeared from the flask's surface. The effect induced by DDT and DDE was again dose d e p e n d e n t but weaker. The lowest concentrations (12 ppm) of DDT and (11 ppm) DDE had little or no effect, and only 49 ppm (DDT) and 44 ppm (DDE) shifted the maxima of the curves to the left. For both c o m p o u n d s the highest concentrations, 81 ppm (DDT) and 88 ppm (DDE) exerted a considerable lethal effect, and the distribution curves were broadened (Fig. 2A and C). DDA inhibited c o l o n y growth much less than the other 3 compounds. Even at the highest c o n c e n t r a t i o n of 100 ppm a distinct m a x i m u m of the distribution curve can be seen (Fig. 2D). The c o n t i n u o u s presence of DDT (8 ppm) in the medium over 3 m ont hs did n o t result in an altered proliferation rate in cultures or colonies. A 4-day treatm e n t of these cells with DDT (49 ppm) caused qualitatively and quantitatively the same effects as shown by cells w i t h o u t pre-treatment, also on the chromosoma] level. This indicates that the sensitivity of the cells was n o t altered. For the evaluation of cellular dose-effect relationships in vitro so-called macro-colonies were usually c o u n t e d r a n d o m l y a week after the plating of single cells or even later, depending on the doubling time of the cell line. In m a n y investigations such macro-colonies consisted of m ore than 50 cells, being descendants of the surviving single cells plated. Colonies having less than 50 cells were then classified as micro-colonies (abortive c l o n e s ) r e p r e s e n t i n g the lethally damaged single cells. T r e a t m e n t with chemical or physical agents may increase the percentage of colonies with less than 50 cells by extending the doubling time a n d / o r enhancing the p r o p o r t i o n of dead cells in subsequent generations. By measuring inhibition of c o l o n y proliferation based on t w o or more mechanisms it is normally impossible to distinguish between t hem witho u t pedigree analysis. This holds true for the m a c r o / m i c r o - c o l o n y system as well as for our system where individually marked colonies are analyzed. The dose-dependent toxic action of a c o m p o u n d as the overall effect just mentioned, however, may be seen in our results, even after a few days, namely when the colonies destined to b e c o m e macro-colonies are distinguishable. Because of the necessity for exact cell countings in the colonies our experiments were n o t c o n t i n u e d for m or e than 4 days. Each day the percentage of dead cells (cell-free circles engraved on the out e r surface of the flasks on day 0) could be determined. Knowing f r om previous investigations that most of the colonies having 16 or more cells on day f our will becom e macro-colonies during the n e x t two days, we t her e f or e t o o k this criterion to group our material: macro-colonies /> 16 cells; micro-colonies ~ 16 cells. The results are shown in Table I. The last column in this table shows the percentage of colonies with more than 32 cells. Again DDA exer t e d the weakest, and DDD and DDT the strongest effect which can already be seen in the low concent rat i on range of a b o u t 20 ppm. In the range of 40--50 ppm the shift from macro- to micro-colonies was still more p r o n o u n c e d with DDD and DDT, and the absolute cell mortality was very high after t r e a t m e n t with DDD (75 ppm) (96%) and DDT (81 p p m ) (82%). Though f ur t her experiments have t o be done for establishing complete dose-effect curves and time curves of t r e a tm ent , the data from our experi-
113 TABLE I D O S E - - D E P E N D E N T L E T H A L A N D T O X I C E F F E C T S OF 4 - D A Y T R E A T M E N T W I T H D D T , D D D , D D E A N D D D A AS D E T E R M I N E D BY M A C R O - A N D M I C R O - C O L O N Y D E V E L O P M E N T U N T I L D A Y F O U R . A L L C O M P O U N D S D I S S O L V E D I N 1% D M S O - - M c C O Y M E D I U M Number of p l a t e d cells marked
Cells k i l l e d
Colonies with 1 - - 1 5 cells
(%)
Colonies with more than 15 cells (%)
Colonies of column 4 with more than 32 cells (%)
(%) Control 0% DMSO
400
12.8
17.8
69.5
46.S
Control 1% DMSO
200
14
27
59
34
DDT ppm
12 24 49 81
100 300 100 i00
11 8 16 82
42 45 84 18
47 47 0 0
18 17 0 0
DDD ppm
11 22 45 75
100 100 100 100
17 25 21 96
14 56 78 4
69 26 2 0
59 8 0 0
DDE ppm
11 22 44 88
100 100 200 100
18 7 13 26
11 7 51 74
71 86 37 0
65 79 21 0
DDA ppm
20 41 50 68
100 100 100 100
5 9 3 6
1S 32 59 93
76 59 38 1
61 20 2 0
ments demonstrate for all four c o m p o u n d s a considerable increase of microcolonies when higher concentrations than 4 1 - - 5 0 ppm were used.
Chromosome damage The spontaneous aberration rates in our cell line were 0 . 0 5 - - 0 . 0 7 gaps/cell and 0.02 breaks/cell. A n o m a l o u s c h r o m o s o m e configurations such as exchanges, rings, decentrics, etc. were rare events and did n o t appear at all in the control series of these experiments. To test concentration dependency, the pesticides were added in various amounts to the medium, together with colcemid, for 4 h (Table II). D D T at 12 and 24 ppm did n o t cause measurable effects but 41 ppm significantly enhanced the gap rate and 81 ppm gap and break rates. With D D D at 45 ppm only the gap rate was enhanced, whereas 75 ppm induced higher aberration rates in both classes. DDE had only a moderate effect. With 44 ppm the gap rate was increased significantly, but 88 ppm also induced enhanced break rates, although n o t as great as in the experiments with D D T and DDD. D D A concentrations as high as 100 ppm increased the gap number but n o t the number of breaks. Morever, the increase in gaps was less for D D A than for any of the other c o m p o u n d s . As m e n t i o n e d above, n o configuration anomalkes were observed in the experiments. Treatment of the cells at high concentrations for more than 4 h led to severe
114 TABLE
II
DOSE-DEPENDENT CYTOGENET1C EFFECTS DISSOLVED IN 1% DMSO--MeCOY MEDIUM Pesticide CONC. (ppm) Control a
OF DDT,
Cell n u m b e r
Gaps
DDD,
DDE
AND DDA. ALL COMPOUNDS
Breaks
G a p s p e r cell
B r e a k s p e r cell
800
56
14
0.07
0.02
---
600 500
38 25
7 5
0.06 0.05
0.01 0.01
DDT 4 h
49 81
200 300
49 171
7 34
0.25 0.57
0.04 b 0.11
DDD 4 h
45 75
300 200
89 105
17 26
0.28 0.53
0.06 0.13
DDE 4 h
44 88
200 200
22 75
6 20
0.11 0.35
0.03 b 0.10
DDA 4 h
41 68 100
200 200 200
10 27 41
1 5 5
0.05 b 0.14 0.21
0.005 b 0.03 b 0.03 b
1% DMSO
4h 24 h
a C o n t r o l , cells i n M c C o y m e d i u m w i t h o u t D M S O . b Data not different from DMSO control (P ~ 0.01).
TABLE
III
TIME-DEPENDENT CYTOGENETIC EFFECTS DISSOLVED IN 1% DMSO-McCOY MEDIUM Time
Cell n u m b e r
OF DDT,
DDD,
DDE
AND
DDA.
ALL COMPOUNDS
Gaps
Breaks
G a p s p e r cell
B r e a k s p e r cell
(h) Control a
_
800
56
14
0.07
0.02
1% DMSO
4 24
600 500
38 25
7 5
0.06 0.05
0.01 0.01
DDT 49 ppm
4 12 24 24 + 24
200 200 200 100
49 51 47 52
7 16 15 8
0.25 0.26 0.24 0.52
0.04 b 0.08 0.08 0.08
DDD 45 ppm
4 12 24 24 + 24
300 200 200 100
89 71 63 36
17 17 22 12
0.28 0.36 0.32 0.36
0.06 b 0.09 0.11 0.12
DDE 44 ppm
4 12 24 24 + 24
200 200 200 100
22 37 34 18
6 8 11 10
0.11 0.19 0.17 0.18
0.03 b 0.04 b 0.06 b 0.10
DDA 41 p p m
4 12 24 24 + 24
200 200 200 100
10 26 22 32
1 5 4 0
0.05 b 0.13 0.11 0.32
0.005 b 0.03 b 0.02 b 0
DDT 8 ppm
3 months
200
25
11
0.10 b
0.06 b
a Control, cells in McCoy medium without DMSO. b Data not different from DMSO control (P ~ 0.01).
115 damage t h a t m a y ~)ecome lethal with DDT and DDD. The number of scorable metaphases also dropped drastically. Therefore, extended time experiments were done with the following concentrations only: DDT 49 ppm; DDD 45 ppm; DDE 44 ppm; DDA 41 ppm (Table III). The table shows t h a t more than 4-h t r e a t m e n t with DDT did n o t further enhance the gap rate and only moderately affected the break rate. The same p h e n o m e n o n was observed with DDD and with DDE in the range of 12 to 24 h. This may indicate a time dependency only within the first hours of treatment. The absence of structural anomalies, such as exchanges, rings etc., could be due to the relatively short time from the end of the t r e a t m e n t until the preparation of the chromosomes (at m a x i m u m 24 h). Often such configuration anomalies appear only in the daughter cells of the first or second post-treatment generation. Therefore, we kept the cells in a pesticide-free medium for another 24 h after the end of the 24-h treatment with the compounds. During this additional day in culture our cells normally underwent 1--2 divisions. Again, we did n o t find dicentrics, exchanges, rings etc. No numerical aberrations were observed. The chronic t r e a t m e n t with DDT at 8 ppm did n o t significantly induce altered gap or aberration rates (Table III).
Discussion
Our results demonstrate that DDD and DDT are much more toxic for Chinese hamster cells in culture than are DDE or DDA, the latter being toxic only at the relatively high concentration of more than 68 ppm. The observed formation of vacuoles and granules in the cells during a 4-day treatment with the four pesticides indicates a possible damage to the cell membrane and cytoplasm. In earlier reports an effect of DDT on the protein and nucleic acid synthesis in liver cells in vitro has already been reported [17,33]. A similar influence was f o u n d in experiments with HeLa cells [6]. In other reports cytoplasmic damage has been discussed as an uptake of lipophilic compounds into the lipid bilayer of the membranes (insects [40,41]; insects and mammals [36,37]; rats and m o n k e y s [9] ). Preliminary gas-chromatographic analysis of our material showed that DDT and its lipophilic metabolites were bound to the cells in different quantities. The a m o u n t of DDE f o u n d in the washed cell sediment after centrifugation was double that of DDD, whereas DDT was in between. This again supports the conclusion t h a t the toxicity range from weak to strong is as follows: DDA, DDE, DDT and DDD. This sequence is confirmed in the 4-h experiments. Though our results were obtained from experiments in vitro a similar effect m a y exist in vivo. The action spectrum of DDD, especially, would be worth while evaluating because in our system this c o m p o u n d is more toxic than DDT. On the other hand it is of interest t h a t t r e a t m e n t of our cells for 3 months with DDT (8 ppm) did n o t change their proliferation characteristics or their sensitivity to acute higher doses of DDT. The chromosomal aberration rates remained in the spontaneous range, so a continuous induction and elimination of visible structural a n d / o r numerical aberrations is unlikely, although from our
116 data it cannot be decided whether sub-microscopic damage is induced and eliminated or continuously repaired. Higher concentrations of all four compounds, however, when applied for days or hours, enhanced the gap rate from a given concentration onward. Depending on dose and time of exposure, the break rates were also enhanced by DDT, DDD and DDE. None of the DDA concentrations increased the break rates. It is obvious that treatment time, concentrations in vitro and other parameters may interfere with the interpretation of observed " m u t a g e n i c " effects. We have chosen the lowest concentration of 8 ppm (DDT) in the three-month experim e n t with respect to the mean values of accumulated DDT reported for human fatty tissues, 5--10 ppm; for the whole body of normal healthy man, 4.9 and 5.3 ppm; for the body of a vegetarian, 2.3 ppm; for the body of a heavily exposed man, 50 ppm [12,21,26,30]. Of course, the higher concentrations applied in our acute treatments are n o t f o u n d in man, but they were chosen to secure measurable effects for the comparative studies. Our results on the differential activity of the four compounds are in good correlation with similar data from the literature. Research on the insect metabolism of pesticides shows that at least 300 species have developed more or less effective resistant mechanisms to DDT, mostly transforming DDT to DDE by dehydrochloronation [3, 39]. This DDE is much less active as an insecticide [27], which agrees with our findings in vitro. On the contrary, in higher animals, DDE is described as a rather damaging c o m p o u n d like DDT. In birds, a well established relation exists between the a m o u n t of DDE in the body and the thickness of the egg shells: the more DDE f o u n d in the organisms the thinner the shells [22]. Different cytogenetic effects and results from experiments with host-mediated assays and dominant-lethal tests were mentioned earlier in this paper [4, 35,49]. These data can only partly elucidate the eventual genetic danger exerted by DDT derivatives in mammals and man. Our data show that the lipophilic compounds, especially DDD, may be much more dangerous than DDA. The pesticides are distributed t h r o u g h o u t the whole mammalian body [1]. In liver cells, as well as in bone marrow cells, electron micrographs demonstrate DDT-induced alterations [16,42]. DDD, DDE and DDA are found in the urine and feces of rats after uptake of DDT, proving the formation of the derivatives by metabolism. It has to be assumed t h a t in all cells of an organism, germ cells included, there is a more or less strong direct or indirect damage by pesticides. Under simultaneous or selective chronic exposure to the pesticides, permanent structural and functional anormalities could become stabilized. On the other hand, experiments with DDT have given contradictory results concerning cancerogenicity [14,15,23,31,50]. Our experiments with somatic cells have n o t rendered adequate information on the relevance of such results to germ cells. It would be of interest, therefore, to repeat the cytogenetic experiments in vivo, thereby comparing the reaction of germ cells (spermatogonia, spermatocytes) and bone marrow cells of Chinese hamsters and/or other laboratory mammals. Another important question arises: if more than one pesticide or agent is present simultaneously within an organism, what are their interactions and are they acting in an additive or an overadditive way? We intend to report on such combined effect in a following paper: one of the agents will be a pesticide.
117
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Mutation Research, 40 (1976) 107--118 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
T H E E F F E C T OF INSECTICIDES ON CHINESE HAMSTER CELL C U L T U R E S
ULRIKE MAHR and HERBERT GEORG MILTENBURGER Institu te of Zoology, Technische Hochschule Darmstadt, D-6100 Darmstadt (Germany)
(Received May 21st, 1975) (Revision received November 18th, 1975) (Accepted November 28th, 1975) Summary The effect o f p,p'-isomers of DDT and its derivatives DDD, DDE and DDA on Chinese hamster cells in culture was studied. At different concentrations and various times o f t r e a t m e n t the proliferation rate was inhibited most strongly by DDD and DDT, whereas DDE exhibited a markedly weaker influence. DDA was the least toxic c o m p o u n d of the four. The cytogenetic effects were also different. Again, DDA induced the least damage. Only enhanced gap rates b u t no c h r o m o s o m e breaks were observed. DDE Was more active, and higher break rates occurred. DDD and DDT were by far the m ost damaging compounds, and t h e y raised the gap and break rates markedly. However, no induction of configuration anomalies was f o u n d in any experiment. Chronic t r e a t m e n t of the cells for 3 m o n t h s with DDT at 8 ppm did n o t alter the proliferation rate, the sensitivity to acute t r e a t m e n t with higher DDT concentrations or th e c h r o m o s o m a l aberration rates. The results are discussed in relation to the relevance of differential pesticide effectivity in organs of higher animals and man.
Introduction During the last 20 years a world-wide accumulation of pesticides in the organs o f m a n y species has been observed. Analyses with different test organisms showed th at DDT (dichloro-diphenyl-trichloroethane) and its metabolic derivatives can induce cellular damage. Though short c o n t a c t at concentrations normally present in the e n v i r o n m e n t did n o t p r o d u c e a measurable effect in man, chronic uptake proved t o be toxic [ 1 0 , 1 8 , 2 0 , 2 8 ] . Under long term exposure conditions the half-value times for the elimination of the pesticides are rather long, sometimes 4 years and more [ 1 1 ] . Most of the water-insoluble pesticides exist as micro-suspensions or form aerosols in the atmosphere. In the latter
108 form they fall to the earth with rain, consequently appearing in the terrestrial water circulation and later in food chains [7,51]. At present the uptake of pesticides is still greater than their elimination in most organisms. In cultures of kangaroo rat cells [44] and in other cell systems in vitro a toxic effect has been demonstrated. Gabliks and Friedman [17] found that 50% of HeLa and Chang liver cells die after a 48-h treatment with DDT (100 ppm). Johnson and Weiss [25] described a 50% proliferation inhibition in HeLa- and KB-cell cultures with a nutrient medium containing 25 pg DDA. Chung et al. [5,6] described a decrease in the cell number and an increase in the protein synthesis in HeLa cells with as little as 0.5 ppm DDT. A short treatment of 4.5 h with DDT (125 ppm) influenced the DNA and RNA synthesis of HeLa cells markedly [34], chicken cells became necrotic, and mitotic divisions were inhibited [47]. In another report, chicken cell cultures did not show signs of damage after treatment with DDT at 600 ppm [32]. The accumulation of metabolic derivatives of DDT in the tissues of higher organisms and the continued use of high levels of the insecticide as a consequence of increased levels of resistance had led to concern not only about the toxicological aspects but also about the possibility of mutagenic action by the metabolites [2,3,13,39,43]. The toxicological, physiological and genetical effects of DDT derivates DDD (dichloro-diphenyl-dichloroethane), DDE (dichloro-diphenyl-dichloroethene) and DDA (dichloro-diphenyl-acetate) have been examined in warm-blooded animals as well as in microorganisms [2,8,19,24,45,46]. The kind of damage varies markedly. Cytogenetic investigations on the effect of pesticides have produced inconsistent results. Experiments on Drosophila did not show mutagenicity of DDT [35]. Investigations using p,p' isomers of DDT, DDD, DDE, DDA and DDOM (2,2-bis(p,chlorophenyl)-ethanol(1)), another metabolite of DDT, resulted in a significant mutagenic effect only for DDA and a weak effect for DDT. The other compounds showed no effect [49]. Comparison of the action of p,p' and o,p' isomers of DDT and the metabolites DDD, DDE and DDA demonstrated a higher effectiveness of thep,p' isomers, which produced twice as many chromosomal aberrations as the o,p' isomers. DDT, DDD, DDE and DDA again produced different effects in the dominantlethal test and in the host-mediated assay. With the latter m e t h o d only DDD proved to be mutagenic, whereas none of the four compounds had any effect in the dominant4ethal test [4]. These results underline the c o m m o n experience in so far as the results may differ depending on the test system and the methods applied. Therefore, we felt it would be worth while to compare the way in which the proliferation-inhibiting and cytogenetic effects of DDT, DDD, DDE and DDA apply to mammalian cells in a standardizable system in vitro. We used a quasi-diploid cell line of the Chinese hamster, having 22/23 chromosomes in the stem line. Materials and methods
Cell cultures Cells of the Chinese hamster cell line B14 F28 were cultivated in 15 ml of
109 McCoy's medium [38] prepared in our laboratory, supplemented with 10% calf serum (GIBCO, New York) and Neomycin sulfate (Byk-Gulden, Konstanz) at 100 mg/1. The culture vessels were 180 ml glass bottles. The trypsinized (0.075% trypsin) cells were transferred to T-30 plastic flasks (FALCON, Los Angeles), and each flask was inoculated with 1200--1500 cells in 5 ml medium. After 4 h the cells had sedimented to the b o t t o m of the flask, where they adhered firmly. The flasks were then completely filled with medium and inverted. With a writing diamond, 50 to 100 single cells were encircled on the outer surface of a flask. Thus, within a circle each cell and its progeny could be observed during the following days, and individual colony growth curves could be established. The hanging position of the cells permitted the use of a normal microscope. Also, dead cells, losing contact with the vessel surface, fell to the opposite side (the b o t t o m ) of the flask, thereby disappearing from the microscopic field. Healthy and non-lethally damaged cells in interphase as well as in mitosis adhered firmly to the inner flask surface while "hanging". Comparative studies did n o t reveal any difference in cell multiplication or colony proliferation for "hanging" and "sitting" cultures. Another advantage of our m e t h o d is that only normally attached cells were analyzed. The plating efficiency was therefore consistent from experiment to experiment.
Pesticide treatment The lipophilic compounds DDT (Roth, Karlsruhe), DDD and DDE (Riedel De Haen, Hannover), not soluble in water, are easily dissolved in dimethylsulfoxide (DMSO; Merck, Darmstadt), which in turn is suspendable in water. In a series of experiments it was confirmed that the final concentration of 1% DMSO in the medium had no influence on the pesticide action. The pesticides were used as p,p' isomers mostly in the following concentrations: DDT 12, 24, 49 and 81 ppm; DDD 11, 22, 45 and 75 ppm; DDE 11, 22, 44 and 88 ppm; DDA 20, 41, 50, 68 and 100 ppm. DDA (Riedel De Haen, Hannover), which is water soluble, was also dissolved in DMSO for better comparison -- as to effects -- with the other substances. Owing to formation of many granules and vacuoles in cells after more than 4 h of treatment with the higher concentrations of the 4 substances, one series of experiments was run for only 4 h. When more than 90% of the cells had adhered to the vessel surface and spread out, the treatment was started, so that •the cells, but not their progeny, were treated with the pesticide. The pesticide medium was replaced by normal medium directly after the treatment time. In another series of experiments the pesticide was present in the medium for 4 days, so that the cells and their progeny were treated over the whole experimental time. Only m e d i u m concentrations in the range of 40--50 ppm were used. In a chronic treatment, the medium of the stock cultures continuously contained DDT at 8 ppm for a period of 3 months, a dose that occurs in mammals and man by accumulation over m a n y years [20,26,29,30]. These cells were then treated with DDT at 49 ppm for 4 days to test whether changes in resistance or sensitivity would occur.
Evaluation of effects The colony sizes, determined by counting the number of cells each day, were
110 grouped into classes of the theoretically possible cell numbers per generation: 1st generation, 2 cells; 2nd generation, 4 cells; 3rd generation, 8 cells etc. From this colony size, distribution curves were established referring to individual colonies and to the mean cell number out of all colonies of an experiment. The cell counts were done daily from day 0 (day of plating) to day 4, and the colonies were classified as those with 1, 2, 3--4, 5--8, 9--16 cells etc. Each value in the distribution curves represents the percentage out of at least 100 colonies. For the evaluation of the cytogenetic effect the metaphase cells were accumulated by 0.068 pg colcemid per ml medium (CIBA, Basel) given 4 h before chromosome preparation, followed by the normal air-drying procedure. Per dose and time at least 100 metaphases were evaluated on coded slides for structural aberrations: chromatid gaps (unstained area in a chromatid being smaller than a chromatid diameter or equal to it), chromatid breaks (opening in the chromatid being greater than the diameter or the chromatid piece was dislocated), including terminal deletions. As none of the four compounds induced increased aberration rates for rings, exchanges, dicentrics etc., such data are omitted from the following description. From the number of chromosomes in all cells scored having 19--25 chromosomes, mean chromosome distribution curves were established for evaluating possible variations of the numerical aberration rates. The cells were treated with different pesticide concentrations and for various exposure times (Tables II and III). For better comparison of an eventual c o m p o u n d action at exposure times of more than 4 h, nearly equal concentrations were tested. The ×2 test was used for statistical analysis. All data were regarded as different from the control when P ~ 0.01. Results
Colony damage The 4-h treatment revealed a differential effect of the four compounds on proliferation characteristics of the colonies during the 4 days after the start of the experiment. DDD-treated cells were damaged most. The effect could still be seen clearly on the 4th day, especially when the highest concentration of 75 ppm was used, even though the medium with the pesticide was replaced by normal medium directly after the 4-h treatment (Fig. 1A). The distribution curve of colony sizes was therefore shifted towards smaller cell numbers, and a m a x i m u m was n o t clearly formed (Fig. 1B). A b o u t 65% of the colonies consisted of not more than 8 cells, and 25% of the cells were lethally damaged as measured by counting the cell-free circles engraved on the outer surface of the flasks. A dose-dependent inhibitory effect on colony growth was also induced with the 45 and 22 ppm concentrations of DDD, but 11 ppm was ineffective. The high doses of DDT (81 ppm) and DDE (88 ppm) influenced the colony growth rate as well but n o t as much, and DDA (100 ppm) did not produce any effect. The medium and low concentrations of these compounds had only weak or no effect. Fig. 2 shows the results of the 4-day treatment presented as colony distribution curves. Again DDD was most effective, inducing a heavy proliferation inhibition. DDD (75 ~pm) killed 96% of the cells and no colony consisted of more than 4
111
100 >_ 60
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4 DAY
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B
q Z
o 30 o
2O
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8 32 1~8 CELLS/COLONY
512
Fig. 1. D D D , 4 - h t r e a t m e n t . ( A ) C o l o n y g r o w t h c u r v e s ; e a c h p o i n t r e p r e s e n t s t h e m e a n o f cells in a t l e a s t 1 0 0 c o l o n i e s . (B) C o l o n y size d i s t r i b u t i o n c u r v e s . A b s c i s s a : cells p e r c o l o n y in l o g a r i t h m i c scale. (o ©) c o n t r o l ; (¢ -~) 7 5 p p m .
m
,
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3C
20 10
'2'
"
8
:T2
lzB-
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v 2 CELLS/COLONY
8
~
128 v
512
Fig. 2. C o l o n y size d i s t r i b u t i o n c u r v e s ; 4 - d a y t r e a t m e n t . A b s c i s s a : cells p e r c o l o n y in l o g a r i t h m i c scale. (o o) c o n t r o l ; (o I ) p p m D D T 1 2 ; D D D 1 1 ; D D E 1 1 ; D D A 2 0 ; (4 4) p p m D D T 2 4 ; D D D 2 2 ; D D E 2 2 ; D D A 4 1 ; (m m) p p m D D T 4 9 ; D D D 4 5 ; D D E 4 4 ; D D A 5 0 ; (0 ,) ppm DDT 8 1 ; D D D 7 5 ; D D E 8 8 ; D D A 6 8 ; ([] D) p p m D D A 1 0 0 .
112
cells. This ef f ect is d e m o n s t r a t e d by the colony size distribution curves where the maxima are drastically shifted towards the smaller cell numbers (Fig. 2B). Even after one day, DDD (75 ppm) killed more than 40% of the cells, which then disappeared from the flask's surface. The effect induced by DDT and DDE was again dose d e p e n d e n t but weaker. The lowest concentrations (12 ppm) of DDT and (11 ppm) DDE had little or no effect, and only 49 ppm (DDT) and 44 ppm (DDE) shifted the maxima of the curves to the left. For both c o m p o u n d s the highest concentrations, 81 ppm (DDT) and 88 ppm (DDE) exerted a considerable lethal effect, and the distribution curves were broadened (Fig. 2A and C). DDA inhibited c o l o n y growth much less than the other 3 compounds. Even at the highest c o n c e n t r a t i o n of 100 ppm a distinct m a x i m u m of the distribution curve can be seen (Fig. 2D). The c o n t i n u o u s presence of DDT (8 ppm) in the medium over 3 m ont hs did n o t result in an altered proliferation rate in cultures or colonies. A 4-day treatm e n t of these cells with DDT (49 ppm) caused qualitatively and quantitatively the same effects as shown by cells w i t h o u t pre-treatment, also on the chromosoma] level. This indicates that the sensitivity of the cells was n o t altered. For the evaluation of cellular dose-effect relationships in vitro so-called macro-colonies were usually c o u n t e d r a n d o m l y a week after the plating of single cells or even later, depending on the doubling time of the cell line. In m a n y investigations such macro-colonies consisted of m ore than 50 cells, being descendants of the surviving single cells plated. Colonies having less than 50 cells were then classified as micro-colonies (abortive c l o n e s ) r e p r e s e n t i n g the lethally damaged single cells. T r e a t m e n t with chemical or physical agents may increase the percentage of colonies with less than 50 cells by extending the doubling time a n d / o r enhancing the p r o p o r t i o n of dead cells in subsequent generations. By measuring inhibition of c o l o n y proliferation based on t w o or more mechanisms it is normally impossible to distinguish between t hem witho u t pedigree analysis. This holds true for the m a c r o / m i c r o - c o l o n y system as well as for our system where individually marked colonies are analyzed. The dose-dependent toxic action of a c o m p o u n d as the overall effect just mentioned, however, may be seen in our results, even after a few days, namely when the colonies destined to b e c o m e macro-colonies are distinguishable. Because of the necessity for exact cell countings in the colonies our experiments were n o t c o n t i n u e d for m or e than 4 days. Each day the percentage of dead cells (cell-free circles engraved on the out e r surface of the flasks on day 0) could be determined. Knowing f r om previous investigations that most of the colonies having 16 or more cells on day f our will becom e macro-colonies during the n e x t two days, we t her e f or e t o o k this criterion to group our material: macro-colonies /> 16 cells; micro-colonies ~ 16 cells. The results are shown in Table I. The last column in this table shows the percentage of colonies with more than 32 cells. Again DDA exer t e d the weakest, and DDD and DDT the strongest effect which can already be seen in the low concent rat i on range of a b o u t 20 ppm. In the range of 40--50 ppm the shift from macro- to micro-colonies was still more p r o n o u n c e d with DDD and DDT, and the absolute cell mortality was very high after t r e a t m e n t with DDD (75 ppm) (96%) and DDT (81 p p m ) (82%). Though f ur t her experiments have t o be done for establishing complete dose-effect curves and time curves of t r e a tm ent , the data from our experi-
113 TABLE I D O S E - - D E P E N D E N T L E T H A L A N D T O X I C E F F E C T S OF 4 - D A Y T R E A T M E N T W I T H D D T , D D D , D D E A N D D D A AS D E T E R M I N E D BY M A C R O - A N D M I C R O - C O L O N Y D E V E L O P M E N T U N T I L D A Y F O U R . A L L C O M P O U N D S D I S S O L V E D I N 1% D M S O - - M c C O Y M E D I U M Number of p l a t e d cells marked
Cells k i l l e d
Colonies with 1 - - 1 5 cells
(%)
Colonies with more than 15 cells (%)
Colonies of column 4 with more than 32 cells (%)
(%) Control 0% DMSO
400
12.8
17.8
69.5
46.S
Control 1% DMSO
200
14
27
59
34
DDT ppm
12 24 49 81
100 300 100 i00
11 8 16 82
42 45 84 18
47 47 0 0
18 17 0 0
DDD ppm
11 22 45 75
100 100 100 100
17 25 21 96
14 56 78 4
69 26 2 0
59 8 0 0
DDE ppm
11 22 44 88
100 100 200 100
18 7 13 26
11 7 51 74
71 86 37 0
65 79 21 0
DDA ppm
20 41 50 68
100 100 100 100
5 9 3 6
1S 32 59 93
76 59 38 1
61 20 2 0
ments demonstrate for all four c o m p o u n d s a considerable increase of microcolonies when higher concentrations than 4 1 - - 5 0 ppm were used.
Chromosome damage The spontaneous aberration rates in our cell line were 0 . 0 5 - - 0 . 0 7 gaps/cell and 0.02 breaks/cell. A n o m a l o u s c h r o m o s o m e configurations such as exchanges, rings, decentrics, etc. were rare events and did n o t appear at all in the control series of these experiments. To test concentration dependency, the pesticides were added in various amounts to the medium, together with colcemid, for 4 h (Table II). D D T at 12 and 24 ppm did n o t cause measurable effects but 41 ppm significantly enhanced the gap rate and 81 ppm gap and break rates. With D D D at 45 ppm only the gap rate was enhanced, whereas 75 ppm induced higher aberration rates in both classes. DDE had only a moderate effect. With 44 ppm the gap rate was increased significantly, but 88 ppm also induced enhanced break rates, although n o t as great as in the experiments with D D T and DDD. D D A concentrations as high as 100 ppm increased the gap number but n o t the number of breaks. Morever, the increase in gaps was less for D D A than for any of the other c o m p o u n d s . As m e n t i o n e d above, n o configuration anomalkes were observed in the experiments. Treatment of the cells at high concentrations for more than 4 h led to severe
114 TABLE
II
DOSE-DEPENDENT CYTOGENET1C EFFECTS DISSOLVED IN 1% DMSO--MeCOY MEDIUM Pesticide CONC. (ppm) Control a
OF DDT,
Cell n u m b e r
Gaps
DDD,
DDE
AND DDA. ALL COMPOUNDS
Breaks
G a p s p e r cell
B r e a k s p e r cell
800
56
14
0.07
0.02
---
600 500
38 25
7 5
0.06 0.05
0.01 0.01
DDT 4 h
49 81
200 300
49 171
7 34
0.25 0.57
0.04 b 0.11
DDD 4 h
45 75
300 200
89 105
17 26
0.28 0.53
0.06 0.13
DDE 4 h
44 88
200 200
22 75
6 20
0.11 0.35
0.03 b 0.10
DDA 4 h
41 68 100
200 200 200
10 27 41
1 5 5
0.05 b 0.14 0.21
0.005 b 0.03 b 0.03 b
1% DMSO
4h 24 h
a C o n t r o l , cells i n M c C o y m e d i u m w i t h o u t D M S O . b Data not different from DMSO control (P ~ 0.01).
TABLE
III
TIME-DEPENDENT CYTOGENETIC EFFECTS DISSOLVED IN 1% DMSO-McCOY MEDIUM Time
Cell n u m b e r
OF DDT,
DDD,
DDE
AND
DDA.
ALL COMPOUNDS
Gaps
Breaks
G a p s p e r cell
B r e a k s p e r cell
(h) Control a
_
800
56
14
0.07
0.02
1% DMSO
4 24
600 500
38 25
7 5
0.06 0.05
0.01 0.01
DDT 49 ppm
4 12 24 24 + 24
200 200 200 100
49 51 47 52
7 16 15 8
0.25 0.26 0.24 0.52
0.04 b 0.08 0.08 0.08
DDD 45 ppm
4 12 24 24 + 24
300 200 200 100
89 71 63 36
17 17 22 12
0.28 0.36 0.32 0.36
0.06 b 0.09 0.11 0.12
DDE 44 ppm
4 12 24 24 + 24
200 200 200 100
22 37 34 18
6 8 11 10
0.11 0.19 0.17 0.18
0.03 b 0.04 b 0.06 b 0.10
DDA 41 p p m
4 12 24 24 + 24
200 200 200 100
10 26 22 32
1 5 4 0
0.05 b 0.13 0.11 0.32
0.005 b 0.03 b 0.02 b 0
DDT 8 ppm
3 months
200
25
11
0.10 b
0.06 b
a Control, cells in McCoy medium without DMSO. b Data not different from DMSO control (P ~ 0.01).
115 damage t h a t m a y ~)ecome lethal with DDT and DDD. The number of scorable metaphases also dropped drastically. Therefore, extended time experiments were done with the following concentrations only: DDT 49 ppm; DDD 45 ppm; DDE 44 ppm; DDA 41 ppm (Table III). The table shows t h a t more than 4-h t r e a t m e n t with DDT did n o t further enhance the gap rate and only moderately affected the break rate. The same p h e n o m e n o n was observed with DDD and with DDE in the range of 12 to 24 h. This may indicate a time dependency only within the first hours of treatment. The absence of structural anomalies, such as exchanges, rings etc., could be due to the relatively short time from the end of the t r e a t m e n t until the preparation of the chromosomes (at m a x i m u m 24 h). Often such configuration anomalies appear only in the daughter cells of the first or second post-treatment generation. Therefore, we kept the cells in a pesticide-free medium for another 24 h after the end of the 24-h treatment with the compounds. During this additional day in culture our cells normally underwent 1--2 divisions. Again, we did n o t find dicentrics, exchanges, rings etc. No numerical aberrations were observed. The chronic t r e a t m e n t with DDT at 8 ppm did n o t significantly induce altered gap or aberration rates (Table III).
Discussion
Our results demonstrate that DDD and DDT are much more toxic for Chinese hamster cells in culture than are DDE or DDA, the latter being toxic only at the relatively high concentration of more than 68 ppm. The observed formation of vacuoles and granules in the cells during a 4-day treatment with the four pesticides indicates a possible damage to the cell membrane and cytoplasm. In earlier reports an effect of DDT on the protein and nucleic acid synthesis in liver cells in vitro has already been reported [17,33]. A similar influence was f o u n d in experiments with HeLa cells [6]. In other reports cytoplasmic damage has been discussed as an uptake of lipophilic compounds into the lipid bilayer of the membranes (insects [40,41]; insects and mammals [36,37]; rats and m o n k e y s [9] ). Preliminary gas-chromatographic analysis of our material showed that DDT and its lipophilic metabolites were bound to the cells in different quantities. The a m o u n t of DDE f o u n d in the washed cell sediment after centrifugation was double that of DDD, whereas DDT was in between. This again supports the conclusion t h a t the toxicity range from weak to strong is as follows: DDA, DDE, DDT and DDD. This sequence is confirmed in the 4-h experiments. Though our results were obtained from experiments in vitro a similar effect m a y exist in vivo. The action spectrum of DDD, especially, would be worth while evaluating because in our system this c o m p o u n d is more toxic than DDT. On the other hand it is of interest t h a t t r e a t m e n t of our cells for 3 months with DDT (8 ppm) did n o t change their proliferation characteristics or their sensitivity to acute higher doses of DDT. The chromosomal aberration rates remained in the spontaneous range, so a continuous induction and elimination of visible structural a n d / o r numerical aberrations is unlikely, although from our
116 data it cannot be decided whether sub-microscopic damage is induced and eliminated or continuously repaired. Higher concentrations of all four compounds, however, when applied for days or hours, enhanced the gap rate from a given concentration onward. Depending on dose and time of exposure, the break rates were also enhanced by DDT, DDD and DDE. None of the DDA concentrations increased the break rates. It is obvious that treatment time, concentrations in vitro and other parameters may interfere with the interpretation of observed " m u t a g e n i c " effects. We have chosen the lowest concentration of 8 ppm (DDT) in the three-month experim e n t with respect to the mean values of accumulated DDT reported for human fatty tissues, 5--10 ppm; for the whole body of normal healthy man, 4.9 and 5.3 ppm; for the body of a vegetarian, 2.3 ppm; for the body of a heavily exposed man, 50 ppm [12,21,26,30]. Of course, the higher concentrations applied in our acute treatments are n o t f o u n d in man, but they were chosen to secure measurable effects for the comparative studies. Our results on the differential activity of the four compounds are in good correlation with similar data from the literature. Research on the insect metabolism of pesticides shows that at least 300 species have developed more or less effective resistant mechanisms to DDT, mostly transforming DDT to DDE by dehydrochloronation [3, 39]. This DDE is much less active as an insecticide [27], which agrees with our findings in vitro. On the contrary, in higher animals, DDE is described as a rather damaging c o m p o u n d like DDT. In birds, a well established relation exists between the a m o u n t of DDE in the body and the thickness of the egg shells: the more DDE f o u n d in the organisms the thinner the shells [22]. Different cytogenetic effects and results from experiments with host-mediated assays and dominant-lethal tests were mentioned earlier in this paper [4, 35,49]. These data can only partly elucidate the eventual genetic danger exerted by DDT derivatives in mammals and man. Our data show that the lipophilic compounds, especially DDD, may be much more dangerous than DDA. The pesticides are distributed t h r o u g h o u t the whole mammalian body [1]. In liver cells, as well as in bone marrow cells, electron micrographs demonstrate DDT-induced alterations [16,42]. DDD, DDE and DDA are found in the urine and feces of rats after uptake of DDT, proving the formation of the derivatives by metabolism. It has to be assumed t h a t in all cells of an organism, germ cells included, there is a more or less strong direct or indirect damage by pesticides. Under simultaneous or selective chronic exposure to the pesticides, permanent structural and functional anormalities could become stabilized. On the other hand, experiments with DDT have given contradictory results concerning cancerogenicity [14,15,23,31,50]. Our experiments with somatic cells have n o t rendered adequate information on the relevance of such results to germ cells. It would be of interest, therefore, to repeat the cytogenetic experiments in vivo, thereby comparing the reaction of germ cells (spermatogonia, spermatocytes) and bone marrow cells of Chinese hamsters and/or other laboratory mammals. Another important question arises: if more than one pesticide or agent is present simultaneously within an organism, what are their interactions and are they acting in an additive or an overadditive way? We intend to report on such combined effect in a following paper: one of the agents will be a pesticide.
117
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