Mutation Research, 280 (1992) 187-193 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1218/92/$05.00
187
MUTGEN 01807
The effect of y-irradiation on the toxicity of malathion in V79 Chinese hamster cells and Molt-4 human lymphocytes J.G. Szekely, M. Goodwin and S. Delaney Radiation Applications Research Branch, AECL Research, Whiteshell Laboratories, Pinawa, Manitoba ROE 1LO, Canada (Received 28 October 1991) (Revision received 20 March 1992) (Accepted 27 March 1992) Keywords: Malathion; Sister-chromatid exchange; Micronuclei; Food irradiation; Pesticide; Polyploid
Summary There is growing interest in the irradiation of food and agricultural products for insect disinfestation, sprout inhibition, delayed ripening and the reduction of microbiological loads. Extensive research has been done on this process, and irradiation to a maximum dose of 10 kGy is recognized as safe by national and international regulatory agencies. The question has been raised, however, whether irradiation of pesticide residues might produce radiation products that were more toxic or less toxic than the original pesticide. To address this question, we observed the effects of 10 kGy of y-radiation on malathion as measured by sister-chromatid exchange (SCE), micronuclei formation, cell survival, growth rate and polyploid formation. We found no significant differences between the effects of irradiated and unirradiated malathion on any of these end-points. Polyploid formation was the most dramatic effect of both irradiated and control malathion on V79 Chinese hamster cells. Cell survival, polyploid formation and growth rate were slightly better in cells treated with irradiated malathion. In Molt-4 human lymphocyte ceils, micronuclei formation was not affected by irradiated or unirradiated malathion. Compared to malathion alone, the lack of such biological effects indicates that none of the presumed radiation-induced breakdown products increased or decreased the endpoints studied. The number of SCE was consistently, but not significantly, higher in the cells treated with irradiated malathion. There were no significant differences in cell survival or micronucleus formation in the human lymphocyte cell line Molt-4 treated with irradiated or control malathion. Thus, the irradiation of the pesticide malathion to 10 kGy, a recommended upper dose for most food irradiations, does not significantly alter its toxicity in these in vitro systems.
The irradiation of fruit and vegetables has been used for insect disinfestation, shelf-life extension and manipulation of the ripening time.
Correspondence: Dr. Joseph G. Szekely, Radiation Applications Research Branch, AECL Research, Whiteshell Laboratories, Pinawa, Manitoba ROE 1L0, Canada.
Some people have raised the concern that any pesticide residue on the surface of the fruit or vegetable might become more toxic after exposure to ionizing radiation (Hansard, 1990). This objection can be answered by considering the low hazard of the original pesticide residue and the small amount of radiolytic product ( ~ 300 m g / k g after 10 kGy) expected from irradiation of the residue (Her Majesty's Stationary Office, 1986).
188
A recent review by Lepine (1991) summarized the available data in the literature about the toxicity of the degradation products of pesticides upon treatment with ionizing radiation. He concluded that it is difficult to draw conclusions about the extent of degradation, the nature of the end-products and the overall toxicological effects of radiation on these compounds because of the small number of studies in the literature. Experimental work, therefore, is needed to verify that there is indeed no unexpected hazard. To study this problem we have looked at the genetic toxicity of one pesticide, the organophosphorus insecticide malathion. It was selected because it is one of the most widely employed pesticides in the world, used to combat beetles, aphids and other pests, and has been found as a residue on some fruit and vegetables. Malathion and other organophosphorus insecticides are useful because they are less toxic and less persistent in the environment than organochlorines. Malathion acts through its effects on the cholinesterase enzyme system (Marquis, 1989). The opposite question is also of interest: is any residual pesticide on a food product de-activated and rendered useless if the food is subsequently irradiated? This is potentially important in the long-term storage of items such as grain, where pesticide may be used to eliminate a persistent insect infestation in storage, and irradiation may be used to effect disinfestation at a terminal elevator. Lippold et al. (1969) have looked at the radiation-induced degradation of malathion in hexane and concluded that a 50-kGy exposure reduced its effectiveness by 71%. In another study, however, Cogburn and Mahany (1969) found that neither the toxicity nor degradation was affected by radiation from 0.25 to 40 kGy. Although malathion can be acutely toxic at high doses, it has a relatively low mammalian toxicity: acute oral LDs0 for male and female rats is 1000 m g / k g and 1375 m g / k g , respectively (The Merck Index, 1989). Malathion has been in widespread use for many years and there are numerous reports that it is not genotoxic or carcinogenic (Carcinogen Program NCI, 1977; Huang, 1973; Shirasu et al., 1976; Wild, 1975; Wong et al., 1989). There are, however, other reports that it or some of its degradation products and impuri-
ties have genotoxic properties (Chen et al., 1981. 1982; Herath et aI., 1989; Nicholas et al., 1979: Nishio and Uyeki, 1981; Garry ct al., 1990). Although the use of malathion is widespread, malathion residues are not a major problem. In Canada, for example, from 1978 to 1986, 10% of the Ontario apple crop was treated with malathion; however, none of the apples sampled had a detectable malathion residue present (Frank ct al., 1989). In most vegetable samples tested by Frank et al. (1987), pesticide residues were below detection limits and most of the positive findings were a fraction (1% to 20%) of the maximum residue limit permitted by the Canadian Food and Drugs Act and Regulations. Malathion is routinely monitored and has been found on some food samples in the United States; however, malathion is not on the Environmental Protection Agency's list of potentially oncogenic pesticides and it is not considered to be a major residue problem (Fan and Jackson, 1989). Materials and methods
Malathion preparation and radiation conditions Pure ( > 99%) malathion, Pestanal T M analytical standard grade (Chromatographic Specialties), was irradiated in an A E C L G a m m a Cell 220 at a dose rate of ~ 164 G y / m i n at 0°C. The irradiated and unirradiated samples were dissolved in ethanol ( E t O H ) at a concentration of 300 ~ g / m l for the stock solutions. The stock solutions were held at 4°C for 1 d before being used in cell survival or genetic toxicity tests so all experiments could start at the same time after irradiation, regardless of dose. In preliminary experiments, not reported here, practical (95%) grade malathion (Pfaltz and Bauer) was used with similar results. Cell culture V79 Chinese hamster cells were maintained at 37°C, with 2% CO 2, in 30 ml of H a m ' s F12 media (HEPES) with 10% fetal bovine serum (FBS) in 75-cm 2 flasks. The cells were set up at 5 × 104 cells per flask and subcultured every 3 - 4 d. Under these conditions the doubling time of the V79 cells was - 11 h. Molt-4 human lymphocyte cells were maintained at 37°C with 5% CO 2 in 30 ml
189 of RPMI-1630 media with 10% FBS. The cells were set up at 105 cells/ml in 75-cm z flasks and subcultured every 3 - 4 d. The Molt-4 cells doubled approximately every 24 h.
stored in the dark until analysed. The SCE were counted with a 100 × oil objective with an Olympus BH-2 microscope.
Micronuclei formation Sister-chromatid exchange On the day before the experiment, 75-cm 2 flasks were inoculated with 3 × 105 V79 cells in 30 ml of media containing 10% FBS. On the following day the appropriate concentration of the stock irradiated or unirradiated malathion and 1 x 10 .5 moles/1 5-bromodeoxyuridine (BrdU) were added (total E t O H concentration was not more than 0.1%) to the cultures. After a 20-h incubation period, 0.1 txg/ml of colcemid was added and the cells were incubated for an additional 2 h. The flasks then were shaken to dislodge mitotic cells. The media with mitotic cells was collected and the cells were pelleted. The cell pellet was carefully resuspended in 5 ml of 0.56% KC1 at 37°C and incubated at 37°C for 15 min. 5 ml of m e t h a n o l ( M e O H ) - a c e t i c acid (3:1) fixative was added and the cells were pelleted. The cells were washed by decanting the supernatant and resuspending the pellet in ~ 5 ml of M e O H : a c e t i c acid for 10 min on ice. The pellet was washed again and then resuspended in 0.2-0.5 ml fresh M e O H : a c e t i c acid. The fixed cell suspension was kept refrigerated overnight. Slides were made by adding a drop of cell suspension and a drop of fixative or M e O H , depending upon the degree of chromosome condensation present, on a washed slide and drying it over a flame. Sister chromatids were differentiated using the method of Goto et al. (1978). Briefly described, the slides were treated in the dark with 10 5 m o l e s / l Hoechst 33258 dye in distilled water for 15 min at room temperature, rinsed in distilled water and air-dried for 2 h. A drop of McIlvaine's buffer (pH 8) was added to the slide, and a coverslip was added and sealed with nail varnish to avoid evaporation. The slides were exposed to the light from two Sylvania-350 blacklight 20-W lamps at 50°C for 15 rain. The cells were stained in 2% Giemsa solution in 0.01 m o l e s / l phosphate buffer (pH 6.8) for 15 min at room temperature, and washed 1 x in buffer and 3 × in distilled water. Coverslips were mounted permanently with Permount TM. The slides were
Molt-4 cells were set up at 5 x 104 c e l l / m l one day before the experiment began. The malathion, irradiated or unirradiated, was added with 3.0 /xg/ml of cytochalasin B and the cells were cultured for 24 h. The cells were pelleted, resuspended for 8 rain in 0.56% KC1 at 37°C, pelleted again and resuspended in fresh 0.56% KCI. Drops of the cell suspension were allowed to run down the slide and dry. The slides were fixed in 100% methanol for 3 min and air-dried. The slides were stained with acridine orange by the method of Hayashi et al. (1983). Briefly, the acridine orange stock solution was 1% in distilled water. Slides were stained for 3 rain in 1 part acridine orange stock and 9 parts M / 1 5 phosphate buffer (pH 6.0) followed by three 3-min rinses in M / 1 5 buffer. The slides were viewed in an Olympus BH-2 microscope with an incident-light fluorescence attachment. Binucleated cells were counted for the presence of micronuclei.
Cellular toxicity V79 Chinese hamster cells were plated at 300 cells/60-mm culture dish in 2 ml of F12 media plus 10% FBS and left to attach for 4 h. The appropriate concentration of malathion was added in 3 ml of medium to give a final volume of 5 ml/dish. The dishes were incubated at 37°C and 2% CO 2 for 6 d. The colonies were stained with 1% methylene blue and counted to determine relative survival. Each dose point was plated with four replicates. Results and discussion
Loss of colony formation is a standard measure of reproductive death caused by an exposure to a chemical or physical insult. The first test was to determine the toxicity of malathion in the V79 cell system. The cells were grown for 7 d in the presence of varying concentrations of irradiated and unirradiated malathion. As seen in Fig. 1, although the unirradiated sample had a slightly lower survival level at each dose point, the effect
190
2-
~n
Fig. 1. T h e S C E s f o u n d in a n u m b e r o f r e p e a t e x p e r i m e n t s a r e s h o w n in T a b l e 1. T h e S C E v a l u e s w e r e s u b m i t t e d to a s q u a r e r o o t t r a n s f o r m a t i o n to m i n i m i z e f l u c t u a t i o n s in t h e v a r i a n c e and analysed using the SAS program TTEST. T h e d i f f e r e n c e s b e t w e e n cells t r e a t e d w i t h u n i t radiated or irradiated malathion were compared
lib
| "E
TABLE 1
69
THE AVERAGE NUMBER OF S1STER-CHROMATID EXCHANGES PER CELL AFTER A 24-h EXPOSURE TO IRRADIATED AND UNIRRADIATED MALATHION
¢r-
0.1
0.05
2'o
4b
do
go
Expt. No.
Radiation dose (kGy)
Malathion concentration (/xg/ml)
SCE/ cell
Std. error
Number of cells
1
0 0 0 0 0 10 10 10
0~ 0 10 20 30 10 20 30
7.74 7.78 7.93 8.18 8.26 8.02 8.32 8.72
0.35 0.36 0.37 0.37 0.36 0.39 0.45 0.39
50 50 50 50 50 50 50 50
2
0 0 0 0 10 10
0 ~' 0 20 30 20 30
7.06 7.20 9.32 8.40 9.24 9.00
0.32 0.45 0.62 0.51 0.44 0.51
50 25 25 25 25 25
3
0 0 0 0 0 10 10 10
0" 0 10 20 30 10 20 30
7.80 7.66 8.34 8.59 8.56 8.40 8.38 8.75
0.33 0.38 0.35 0.47 0.45 0.38 0.39 0.96
50 50 50 32 25 50 50 8
4
0 0 0 10 10
0 ~' 0 20 20 30
7.92 8.08 6.76 7.60 9.32
0.34 0.72 0.60 0.46 0.67
50 25 25 25 25
5
0 0 0 0 0 50 50 50
0" 0 10 20 30 10 20 30
8.64 9.42 9.64 9.72 9.78 10.26 10.26 10.62
0.46 0.44 0.50 0.46 0.51 0.40 0.40 0.53
50 50 50 50 50 50 50 50
t6o
Malathion ConcentratTon (pg/mL) Fig. 1. The effect of irradiated (o) and unirradiated (11) malathion on V79 cell survival as measured by colony counting.
o f m a l a t h i o n on cell survival did n o t c h a n g e significantly with irradiation. The curves are shaped like r a d i a t i o n - s u r v i v a l curves. U s i n g t h e s i m p l e m u l t i - t a r g e t m o d e l as f i t t e d by t h e S A S p r o g r a m N L I N , n, t h e e x t r a p o l a t i o n n u m b e r , a n d D 0, t h e l o g - r e d u c t i o n c o n c e n t r a t i o n , v a l u e s o f 2.51 _+ 0.53, 29.0 _+ 3.65 / x g / m l ; a n d 2.93 + 0.51, 29.1 _+ 3.07 / x g / m l w e r e o b t a i n e d for t h e u n i r r a d i a t e d a n d 10-kGy-irradiated malathion samples respectively. E a c h d a t a p o i n t s h o w n in Fig, 1 is t h e average of 4 Expts, each having 4 replicate plates per dose point. One sample of malathion, which was i r r a d i a t e d to a d o s e o f 50 kGy, was also t e s t e d for its e f f e c t o n cell survival: n o s i g n i f i c a n t difference between the 50-kGy-irradiated sample a n d its u n i r r a d i a t e d c o n t r o l was f o u n d in this single e x p e r i m e n t . T o test if i r r a d i a t i o n h a d any e f f e c t o n t h e g e n e t i c toxicity o f m a l a t h i o n , b o t h S C E a n d mic r o n u c l e i f o r m a t i o n e x p e r i m e n t s w e r e run. T h e d o s e r a n g e was s e l e c t e d f r o m t h e i n f o r m a t i o n g e n e r a t e d in t h e survival e x p e r i m e n t s h o w n in
a Solvent control.
191 TABLE 2
10-
THE E F F E C T OF M A L A T H I O N C O N C E N T R A T I O N ON THE R E P R O D U C T I V E I N D E X (RI) OF V79 CELLS
LU
~8-
t
,
0
$7-
t~o
Malathion Concentration (pg / ml )
Fig. 2. The effect of irradiated ( m ) and unirradiated ( I ) malathion on sister chromatid formation in V79 cells.
by t-test at each dose level: no differences were significant at the p = 0.05 level in any of the 5 Expts. In general, the irradiated samples had a higher number of SCE as seen in Fig. 2, which shows a graphic representation of the data from Expt. 1. The differences in SCE between cells treated with irradiated or unirradiated malathion, however, were within the error bars. When the data of Expts. 1 - 4 were combined and tested by the Duncan's multiple-range test at p = 0.05 using the SAS G L M program, only the 30-/xg/ml irradiated malathion had a SCE value significantly different than the control. The slope calculated by linear regression from the data summarized in Table 1 for SCE versus malathion concentration was not significantly different from zero at the p = 0.05 level. Only the 10 kGy-irradiation malathion had a significant slope, 0.028 _+ 0.011 S C E / ( / x g / m l ) , and it was small. The reproductive index, RI (M~ + 2 M 2 + 3 M 3 ) / ( M 1 + M z + M3), where M 1, M 2 and M 3 are the n u m b e r of cells that have completed one, two or three cell cycles in the treatment time. This parameter, which is a measure of the cellcycle delay induced by the treatment, is accessible in the SCE analysis because the first generation cells are uniformly stained, the second generation cells have one light and one dark chromatid per chromosome, and the third generation cells have an uneven distribution of light and dark chromatids. In this test some malathion was irradiated to a higher dose of 50 kGy. As seen from Table 2,
Radiation dose (kGy)
Malathion concentration (/xg/ml)
RI
Std. error
0
2.09
0.04
0 0 0 0
10 20 30 40
1.96 1.57 1.35 1.12
0.01 0.04 0.02 0.04
10 10 10 10
10 20 30 40
1.96 1.69 1.41 1.17
0.10 0.02 0.09 0.05
50 50 50 50
10 20 30 40
1.93 1.69 1.48 1.08
0.05 0.05 0.05 0.05
0 "
a Solvent control. b Estimate.
malathion addition slows cell growth; however, irradiation did not have a significant effect upon the decline in reproductive index in V79 cells. In our hands the principle toxic effect of malathion was the production of polyploidy. The percentage of polyploid cells increased with malathion dose such that, for exposures over 40 /xg/ml, the large number of polyploid cells made it difficult to count SCEs. Fig. 3 shows the effect of malathion addition on polyploid formation af-
.J
60-
"6 o.
40-
~0-
II 0
10
20
30
40
Malathion Concentration(~g/mL) Fig. 3. The effect of irradiated ( l l ) and unirradiated (e) malathion on polyploid formation in V79 ceils.
192
o 0 0 0
8 6
"8
g .b 2~
4 2
b
2'0
4'0
6'0
8'0
100
120
Concentration of Malathion (pg/mL) Fig. 4. The effect of irradiated (o) and unirradiated ( O ) malathion on micronuclei formation in Molt-4 cells.
ter a 24-h exposure. The unirradiated samples had slightly higher numbers of polyploid cells. Most of the polyploidy was in cells delayed in the first cell cycle after the addition of malathion. Micronuclei formation is another widely used measure of genetic toxicity. A second cell type, Molt-4, derived from human T-lymphocytes, were used with the micronucleus formation assay. The results of micronuclei measurements are seen in Fig. 4. Although there is a fair bit of scatter, the results show that there is no difference between the irradiated and unirradiated malathion samples. A linear-regression-fit of the data of Fig. 4 showed that the slope was not significantly different from zero ( p = 0.05) for both samples. We were able to use higher dose levels with the Molt-4 cells than with the V79 cells since malathion was less toxic in the Molt-4 cells as shown in Table 3. The experimental conditions, however, were different for the Molt-4 cells: cell toxicity was determined by the percent of dead cells and the increase in cell number after 3 d of incubation in the presence of malathion, in contrast to colony counting in V79 cells. The main objective of this study was to determine if irradiation effected the toxicity of the organophosphorus pesticide malathion in selected in vitro tests. We concluded that for the end-points measured, a 10 kGy y-exposure did not alter the toxicity significantly. This contrasts with some very early work, which showed a large decrease in insect toxicity of malathion dissolved in hexane after a radiation exposure (Lippold et al., 1969), but our results are qualitatively in
accord with those obtained by Cogburn and Mahany (1969), who irradiated malathion in a dry state without a solvent. Our experiments showed a slight decrease in cell killing and polyploid formation after a radiation exposure; but none of the changes was statistically significant. Also there was a small, nonsignificant increase in SCEs produced by irradiated as compared to unirradiated malathion. This is probably another manifestation of the small decrease in cell killing and polyploidy in the irradiated malathion. More cells completed a second division in the irradiated malathion and could be counted in the SCE analysis. Most polyploid cells are held at the first mitosis and are not analysed for SCE or micronuclei, but they may still carry chromosomal damage. In general, malathion, at the doses we tested, did not cause SCE or micronuclei formation. The principle effect of malathion was polyploid formation and that was probably the cause of cell killing reported in Fig. 1. There is not much information in the literature that describes this aspect of malathion. It was noted, however, in the study by Chen et al. (1981), where they showed 50% of V79 cells were polyploid in the first cell cycle after exposure to 40 > g / m l of malathion. The polyploid levels we report are higher; this may reflect the high-purity malathion samples we used.
TABLE 3 T O X I C I T Y OF M A L A T H I O N ON MOLT-4 CELLS MEAS U R E D A F T E R 3-d E X P O S U R E Each value is the mean of three Expts. Malathion concentration (>g/ml)
D e a d cells (%)
Cell number (10 ~ × c e l l s / m l )
240 120 80 6/) 30 15 7.5 0.0 " 0.0
49 30 12 2.8 3.0 1.6 1.0 1.6 1.1
0.19 0.14 0.36 0.43 0.67 0.75 1.1 0.93 1/.98
a Solvent control.
193 I n c o n c l u s i o n , m a l a t h i o n p r o d u c e d a s m a l l inc r e a s e i n s i s t e r - c h r o m a t i d e x c h a n g e in V 7 9 cells. The main effect of malathion was polyploidy form a t i o n . I r r a d i a t i o n o f m a l a t h i o n d i d n o t a l t e r its t o x i c i t y t o a s i g n i f i c a n t l e v e l in a n y o f t h e t o x i c i t y assays we tested.
References Carcinogen Program NCI (1977) Bioassay of malathion for possible carcinogenicity, Natl. Tech. Inform. Serv., PB-278; 527: 98. Chen, H.H., J.L. Hsueh, S.R. Sirianni and C.C. Huang (1981) Induction of sister-chromatid exchanges and cell cycle delay in cultured mammalian cells treated with eight organophosphorus pesticides, Mutation Res., 88, 307-316. Chen, H.H., S.R. Sirianni and C.C. Huang (1982) Sister chromatid exchanges in Chinese hamster cells treated with seventeen organophosphorus compounds in the presence of a metabolic activation system, Environ. Mutagen., 4, 621-624. Cogburn, R.R., and P.C. Mahany (1969) Effect of gamma radiation on the insecticidal efficiency of malathion deposits on wheat and kraft paper, J. Econ. Entomol., 62, 829-831. Fan, A.M., and J.J. Jackson (1989) Pesticides and food safety, Regul. Toxicol. Pharmacol., 9, 158-174. Frank, R., H.E. Braun and B.D. Ripley (1987) Residues of insecticides, fungicides, and herbicides on Ontario-grown vegetables, 1980-1985, J. Assoc. Anal. Chem., 70, 1081 1086. Frank, R., H.E. Braun and B.D. Ripley (1989) Monitoring Ontario-grown apples for pest control chemicals used in their production, 1978-86, Food Additives and Contaminants, 6, 227-234. Garry, V.F., R.L. Nelson, J. Griffith and M. Harkins (1990) Preparation for human study of pesticide applicators: sister chromatid exchanges and chromosome aberrations in cultured human lymphocytes exposed to selected fumigants, Teratogen. Carcinogen. Mutagen., 10, 21-29. Goto, K., Maeda, Y. Kano and T. Sugiyama (1978) Factors
involved in differential Giemsa-staining of sister chromatids, Chromosoma, 66, 351-359. Hansard (1990) The Parliamentary Debates, House of Lords Official Report No. 1456, Great Britain, pp. 598-560. Hayashi, M., T. Sofuni and M. Ishidate Jr. (1983) An application of acridine orange fluorescent staining to the micronucleus test, Mutation Res., 120, 241-247. Her Majesty's Stationary Office (1986) The safety and wholesomeness of irradiated foods, London, p. 17. Herath, J.F., S.M. Jalal, M.J. Ebertz and J.T. Martsolf (1989) Genotoxicity of the organophosphorus insecticide malathion based on human lymphocytes in culture, Cytologia, 54, 191-195. Huang, C.C. (1973) Effect on growth but not on chromosomes of the mammalian cells after treatment with three organophosphorus insecticides, Proc. Soc. Exp. Biol. Med., 142, 36-40. Lepine, F.L. (1991) Effects of ionizing radiation on pesticides in a food irradiation perspective: A bibliographic review. J. Agric. Food Chem., 38, 2112 2118. Lippold, P.C., J.S. Cleere, L.M. Massey Jr., J.B. Bourke and A.W. Avens (1969) Degradation of insecticides by cobalt-60 gamma radiation, J. Econ. Entomol., 62, 1509-1510. Marquis, J.K. (1989) General toxicology of pesticides in: J.K. Marquid (Ed.), A Guide to General Toxicology, 2nd edn., Karger, Basel, pp. 157-178. Nicholas, A.H., M. Vienne and H. Van Den Berghe (1979) Induction of sister-chromatid exchanges in cultured human cells by an organophosphorus insecticide: malathion, Mutation Res., 67, 167-172. Nishio, A., and E.M. Uyeki (1981) Induction of sister chromatid exchanges in Chinese hamster ovary cells by organophosphate insecticides and their oxygen analogs, J. Toxicol. Environ. Health, 8, 939-946. Shirasu, Y., M. Moriya, K. Kato, A. Furuhashi and T. Kada (1976) Mutagenicity screening of pesticides in the microbial system, Mutation Res., 40, 19-30. The Merck Index 7th edn., S. Budavari (Ed). (1989) Merck, Rahway, N J, p. 5586. Wild, D. (1975) Mutagenicity studies on organophosphorus insecticides, Mutation Res., 32, 133-150. Wong, P.K, C.C. Wai and E. Liong (1989) Comparative study on mutagenicities of organophosphorus insecticides in Salmonella, Chemosphere, 18, 2413-2422.
187
MUTGEN 01807
The effect of y-irradiation on the toxicity of malathion in V79 Chinese hamster cells and Molt-4 human lymphocytes J.G. Szekely, M. Goodwin and S. Delaney Radiation Applications Research Branch, AECL Research, Whiteshell Laboratories, Pinawa, Manitoba ROE 1LO, Canada (Received 28 October 1991) (Revision received 20 March 1992) (Accepted 27 March 1992) Keywords: Malathion; Sister-chromatid exchange; Micronuclei; Food irradiation; Pesticide; Polyploid
Summary There is growing interest in the irradiation of food and agricultural products for insect disinfestation, sprout inhibition, delayed ripening and the reduction of microbiological loads. Extensive research has been done on this process, and irradiation to a maximum dose of 10 kGy is recognized as safe by national and international regulatory agencies. The question has been raised, however, whether irradiation of pesticide residues might produce radiation products that were more toxic or less toxic than the original pesticide. To address this question, we observed the effects of 10 kGy of y-radiation on malathion as measured by sister-chromatid exchange (SCE), micronuclei formation, cell survival, growth rate and polyploid formation. We found no significant differences between the effects of irradiated and unirradiated malathion on any of these end-points. Polyploid formation was the most dramatic effect of both irradiated and control malathion on V79 Chinese hamster cells. Cell survival, polyploid formation and growth rate were slightly better in cells treated with irradiated malathion. In Molt-4 human lymphocyte ceils, micronuclei formation was not affected by irradiated or unirradiated malathion. Compared to malathion alone, the lack of such biological effects indicates that none of the presumed radiation-induced breakdown products increased or decreased the endpoints studied. The number of SCE was consistently, but not significantly, higher in the cells treated with irradiated malathion. There were no significant differences in cell survival or micronucleus formation in the human lymphocyte cell line Molt-4 treated with irradiated or control malathion. Thus, the irradiation of the pesticide malathion to 10 kGy, a recommended upper dose for most food irradiations, does not significantly alter its toxicity in these in vitro systems.
The irradiation of fruit and vegetables has been used for insect disinfestation, shelf-life extension and manipulation of the ripening time.
Correspondence: Dr. Joseph G. Szekely, Radiation Applications Research Branch, AECL Research, Whiteshell Laboratories, Pinawa, Manitoba ROE 1L0, Canada.
Some people have raised the concern that any pesticide residue on the surface of the fruit or vegetable might become more toxic after exposure to ionizing radiation (Hansard, 1990). This objection can be answered by considering the low hazard of the original pesticide residue and the small amount of radiolytic product ( ~ 300 m g / k g after 10 kGy) expected from irradiation of the residue (Her Majesty's Stationary Office, 1986).
188
A recent review by Lepine (1991) summarized the available data in the literature about the toxicity of the degradation products of pesticides upon treatment with ionizing radiation. He concluded that it is difficult to draw conclusions about the extent of degradation, the nature of the end-products and the overall toxicological effects of radiation on these compounds because of the small number of studies in the literature. Experimental work, therefore, is needed to verify that there is indeed no unexpected hazard. To study this problem we have looked at the genetic toxicity of one pesticide, the organophosphorus insecticide malathion. It was selected because it is one of the most widely employed pesticides in the world, used to combat beetles, aphids and other pests, and has been found as a residue on some fruit and vegetables. Malathion and other organophosphorus insecticides are useful because they are less toxic and less persistent in the environment than organochlorines. Malathion acts through its effects on the cholinesterase enzyme system (Marquis, 1989). The opposite question is also of interest: is any residual pesticide on a food product de-activated and rendered useless if the food is subsequently irradiated? This is potentially important in the long-term storage of items such as grain, where pesticide may be used to eliminate a persistent insect infestation in storage, and irradiation may be used to effect disinfestation at a terminal elevator. Lippold et al. (1969) have looked at the radiation-induced degradation of malathion in hexane and concluded that a 50-kGy exposure reduced its effectiveness by 71%. In another study, however, Cogburn and Mahany (1969) found that neither the toxicity nor degradation was affected by radiation from 0.25 to 40 kGy. Although malathion can be acutely toxic at high doses, it has a relatively low mammalian toxicity: acute oral LDs0 for male and female rats is 1000 m g / k g and 1375 m g / k g , respectively (The Merck Index, 1989). Malathion has been in widespread use for many years and there are numerous reports that it is not genotoxic or carcinogenic (Carcinogen Program NCI, 1977; Huang, 1973; Shirasu et al., 1976; Wild, 1975; Wong et al., 1989). There are, however, other reports that it or some of its degradation products and impuri-
ties have genotoxic properties (Chen et al., 1981. 1982; Herath et aI., 1989; Nicholas et al., 1979: Nishio and Uyeki, 1981; Garry ct al., 1990). Although the use of malathion is widespread, malathion residues are not a major problem. In Canada, for example, from 1978 to 1986, 10% of the Ontario apple crop was treated with malathion; however, none of the apples sampled had a detectable malathion residue present (Frank ct al., 1989). In most vegetable samples tested by Frank et al. (1987), pesticide residues were below detection limits and most of the positive findings were a fraction (1% to 20%) of the maximum residue limit permitted by the Canadian Food and Drugs Act and Regulations. Malathion is routinely monitored and has been found on some food samples in the United States; however, malathion is not on the Environmental Protection Agency's list of potentially oncogenic pesticides and it is not considered to be a major residue problem (Fan and Jackson, 1989). Materials and methods
Malathion preparation and radiation conditions Pure ( > 99%) malathion, Pestanal T M analytical standard grade (Chromatographic Specialties), was irradiated in an A E C L G a m m a Cell 220 at a dose rate of ~ 164 G y / m i n at 0°C. The irradiated and unirradiated samples were dissolved in ethanol ( E t O H ) at a concentration of 300 ~ g / m l for the stock solutions. The stock solutions were held at 4°C for 1 d before being used in cell survival or genetic toxicity tests so all experiments could start at the same time after irradiation, regardless of dose. In preliminary experiments, not reported here, practical (95%) grade malathion (Pfaltz and Bauer) was used with similar results. Cell culture V79 Chinese hamster cells were maintained at 37°C, with 2% CO 2, in 30 ml of H a m ' s F12 media (HEPES) with 10% fetal bovine serum (FBS) in 75-cm 2 flasks. The cells were set up at 5 × 104 cells per flask and subcultured every 3 - 4 d. Under these conditions the doubling time of the V79 cells was - 11 h. Molt-4 human lymphocyte cells were maintained at 37°C with 5% CO 2 in 30 ml
189 of RPMI-1630 media with 10% FBS. The cells were set up at 105 cells/ml in 75-cm z flasks and subcultured every 3 - 4 d. The Molt-4 cells doubled approximately every 24 h.
stored in the dark until analysed. The SCE were counted with a 100 × oil objective with an Olympus BH-2 microscope.
Micronuclei formation Sister-chromatid exchange On the day before the experiment, 75-cm 2 flasks were inoculated with 3 × 105 V79 cells in 30 ml of media containing 10% FBS. On the following day the appropriate concentration of the stock irradiated or unirradiated malathion and 1 x 10 .5 moles/1 5-bromodeoxyuridine (BrdU) were added (total E t O H concentration was not more than 0.1%) to the cultures. After a 20-h incubation period, 0.1 txg/ml of colcemid was added and the cells were incubated for an additional 2 h. The flasks then were shaken to dislodge mitotic cells. The media with mitotic cells was collected and the cells were pelleted. The cell pellet was carefully resuspended in 5 ml of 0.56% KC1 at 37°C and incubated at 37°C for 15 min. 5 ml of m e t h a n o l ( M e O H ) - a c e t i c acid (3:1) fixative was added and the cells were pelleted. The cells were washed by decanting the supernatant and resuspending the pellet in ~ 5 ml of M e O H : a c e t i c acid for 10 min on ice. The pellet was washed again and then resuspended in 0.2-0.5 ml fresh M e O H : a c e t i c acid. The fixed cell suspension was kept refrigerated overnight. Slides were made by adding a drop of cell suspension and a drop of fixative or M e O H , depending upon the degree of chromosome condensation present, on a washed slide and drying it over a flame. Sister chromatids were differentiated using the method of Goto et al. (1978). Briefly described, the slides were treated in the dark with 10 5 m o l e s / l Hoechst 33258 dye in distilled water for 15 min at room temperature, rinsed in distilled water and air-dried for 2 h. A drop of McIlvaine's buffer (pH 8) was added to the slide, and a coverslip was added and sealed with nail varnish to avoid evaporation. The slides were exposed to the light from two Sylvania-350 blacklight 20-W lamps at 50°C for 15 rain. The cells were stained in 2% Giemsa solution in 0.01 m o l e s / l phosphate buffer (pH 6.8) for 15 min at room temperature, and washed 1 x in buffer and 3 × in distilled water. Coverslips were mounted permanently with Permount TM. The slides were
Molt-4 cells were set up at 5 x 104 c e l l / m l one day before the experiment began. The malathion, irradiated or unirradiated, was added with 3.0 /xg/ml of cytochalasin B and the cells were cultured for 24 h. The cells were pelleted, resuspended for 8 rain in 0.56% KC1 at 37°C, pelleted again and resuspended in fresh 0.56% KCI. Drops of the cell suspension were allowed to run down the slide and dry. The slides were fixed in 100% methanol for 3 min and air-dried. The slides were stained with acridine orange by the method of Hayashi et al. (1983). Briefly, the acridine orange stock solution was 1% in distilled water. Slides were stained for 3 rain in 1 part acridine orange stock and 9 parts M / 1 5 phosphate buffer (pH 6.0) followed by three 3-min rinses in M / 1 5 buffer. The slides were viewed in an Olympus BH-2 microscope with an incident-light fluorescence attachment. Binucleated cells were counted for the presence of micronuclei.
Cellular toxicity V79 Chinese hamster cells were plated at 300 cells/60-mm culture dish in 2 ml of F12 media plus 10% FBS and left to attach for 4 h. The appropriate concentration of malathion was added in 3 ml of medium to give a final volume of 5 ml/dish. The dishes were incubated at 37°C and 2% CO 2 for 6 d. The colonies were stained with 1% methylene blue and counted to determine relative survival. Each dose point was plated with four replicates. Results and discussion
Loss of colony formation is a standard measure of reproductive death caused by an exposure to a chemical or physical insult. The first test was to determine the toxicity of malathion in the V79 cell system. The cells were grown for 7 d in the presence of varying concentrations of irradiated and unirradiated malathion. As seen in Fig. 1, although the unirradiated sample had a slightly lower survival level at each dose point, the effect
190
2-
~n
Fig. 1. T h e S C E s f o u n d in a n u m b e r o f r e p e a t e x p e r i m e n t s a r e s h o w n in T a b l e 1. T h e S C E v a l u e s w e r e s u b m i t t e d to a s q u a r e r o o t t r a n s f o r m a t i o n to m i n i m i z e f l u c t u a t i o n s in t h e v a r i a n c e and analysed using the SAS program TTEST. T h e d i f f e r e n c e s b e t w e e n cells t r e a t e d w i t h u n i t radiated or irradiated malathion were compared
lib
| "E
TABLE 1
69
THE AVERAGE NUMBER OF S1STER-CHROMATID EXCHANGES PER CELL AFTER A 24-h EXPOSURE TO IRRADIATED AND UNIRRADIATED MALATHION
¢r-
0.1
0.05
2'o
4b
do
go
Expt. No.
Radiation dose (kGy)
Malathion concentration (/xg/ml)
SCE/ cell
Std. error
Number of cells
1
0 0 0 0 0 10 10 10
0~ 0 10 20 30 10 20 30
7.74 7.78 7.93 8.18 8.26 8.02 8.32 8.72
0.35 0.36 0.37 0.37 0.36 0.39 0.45 0.39
50 50 50 50 50 50 50 50
2
0 0 0 0 10 10
0 ~' 0 20 30 20 30
7.06 7.20 9.32 8.40 9.24 9.00
0.32 0.45 0.62 0.51 0.44 0.51
50 25 25 25 25 25
3
0 0 0 0 0 10 10 10
0" 0 10 20 30 10 20 30
7.80 7.66 8.34 8.59 8.56 8.40 8.38 8.75
0.33 0.38 0.35 0.47 0.45 0.38 0.39 0.96
50 50 50 32 25 50 50 8
4
0 0 0 10 10
0 ~' 0 20 20 30
7.92 8.08 6.76 7.60 9.32
0.34 0.72 0.60 0.46 0.67
50 25 25 25 25
5
0 0 0 0 0 50 50 50
0" 0 10 20 30 10 20 30
8.64 9.42 9.64 9.72 9.78 10.26 10.26 10.62
0.46 0.44 0.50 0.46 0.51 0.40 0.40 0.53
50 50 50 50 50 50 50 50
t6o
Malathion ConcentratTon (pg/mL) Fig. 1. The effect of irradiated (o) and unirradiated (11) malathion on V79 cell survival as measured by colony counting.
o f m a l a t h i o n on cell survival did n o t c h a n g e significantly with irradiation. The curves are shaped like r a d i a t i o n - s u r v i v a l curves. U s i n g t h e s i m p l e m u l t i - t a r g e t m o d e l as f i t t e d by t h e S A S p r o g r a m N L I N , n, t h e e x t r a p o l a t i o n n u m b e r , a n d D 0, t h e l o g - r e d u c t i o n c o n c e n t r a t i o n , v a l u e s o f 2.51 _+ 0.53, 29.0 _+ 3.65 / x g / m l ; a n d 2.93 + 0.51, 29.1 _+ 3.07 / x g / m l w e r e o b t a i n e d for t h e u n i r r a d i a t e d a n d 10-kGy-irradiated malathion samples respectively. E a c h d a t a p o i n t s h o w n in Fig, 1 is t h e average of 4 Expts, each having 4 replicate plates per dose point. One sample of malathion, which was i r r a d i a t e d to a d o s e o f 50 kGy, was also t e s t e d for its e f f e c t o n cell survival: n o s i g n i f i c a n t difference between the 50-kGy-irradiated sample a n d its u n i r r a d i a t e d c o n t r o l was f o u n d in this single e x p e r i m e n t . T o test if i r r a d i a t i o n h a d any e f f e c t o n t h e g e n e t i c toxicity o f m a l a t h i o n , b o t h S C E a n d mic r o n u c l e i f o r m a t i o n e x p e r i m e n t s w e r e run. T h e d o s e r a n g e was s e l e c t e d f r o m t h e i n f o r m a t i o n g e n e r a t e d in t h e survival e x p e r i m e n t s h o w n in
a Solvent control.
191 TABLE 2
10-
THE E F F E C T OF M A L A T H I O N C O N C E N T R A T I O N ON THE R E P R O D U C T I V E I N D E X (RI) OF V79 CELLS
LU
~8-
t
,
0
$7-
t~o
Malathion Concentration (pg / ml )
Fig. 2. The effect of irradiated ( m ) and unirradiated ( I ) malathion on sister chromatid formation in V79 cells.
by t-test at each dose level: no differences were significant at the p = 0.05 level in any of the 5 Expts. In general, the irradiated samples had a higher number of SCE as seen in Fig. 2, which shows a graphic representation of the data from Expt. 1. The differences in SCE between cells treated with irradiated or unirradiated malathion, however, were within the error bars. When the data of Expts. 1 - 4 were combined and tested by the Duncan's multiple-range test at p = 0.05 using the SAS G L M program, only the 30-/xg/ml irradiated malathion had a SCE value significantly different than the control. The slope calculated by linear regression from the data summarized in Table 1 for SCE versus malathion concentration was not significantly different from zero at the p = 0.05 level. Only the 10 kGy-irradiation malathion had a significant slope, 0.028 _+ 0.011 S C E / ( / x g / m l ) , and it was small. The reproductive index, RI (M~ + 2 M 2 + 3 M 3 ) / ( M 1 + M z + M3), where M 1, M 2 and M 3 are the n u m b e r of cells that have completed one, two or three cell cycles in the treatment time. This parameter, which is a measure of the cellcycle delay induced by the treatment, is accessible in the SCE analysis because the first generation cells are uniformly stained, the second generation cells have one light and one dark chromatid per chromosome, and the third generation cells have an uneven distribution of light and dark chromatids. In this test some malathion was irradiated to a higher dose of 50 kGy. As seen from Table 2,
Radiation dose (kGy)
Malathion concentration (/xg/ml)
RI
Std. error
0
2.09
0.04
0 0 0 0
10 20 30 40
1.96 1.57 1.35 1.12
0.01 0.04 0.02 0.04
10 10 10 10
10 20 30 40
1.96 1.69 1.41 1.17
0.10 0.02 0.09 0.05
50 50 50 50
10 20 30 40
1.93 1.69 1.48 1.08
0.05 0.05 0.05 0.05
0 "
a Solvent control. b Estimate.
malathion addition slows cell growth; however, irradiation did not have a significant effect upon the decline in reproductive index in V79 cells. In our hands the principle toxic effect of malathion was the production of polyploidy. The percentage of polyploid cells increased with malathion dose such that, for exposures over 40 /xg/ml, the large number of polyploid cells made it difficult to count SCEs. Fig. 3 shows the effect of malathion addition on polyploid formation af-
.J
60-
"6 o.
40-
~0-
II 0
10
20
30
40
Malathion Concentration(~g/mL) Fig. 3. The effect of irradiated ( l l ) and unirradiated (e) malathion on polyploid formation in V79 ceils.
192
o 0 0 0
8 6
"8
g .b 2~
4 2
b
2'0
4'0
6'0
8'0
100
120
Concentration of Malathion (pg/mL) Fig. 4. The effect of irradiated (o) and unirradiated ( O ) malathion on micronuclei formation in Molt-4 cells.
ter a 24-h exposure. The unirradiated samples had slightly higher numbers of polyploid cells. Most of the polyploidy was in cells delayed in the first cell cycle after the addition of malathion. Micronuclei formation is another widely used measure of genetic toxicity. A second cell type, Molt-4, derived from human T-lymphocytes, were used with the micronucleus formation assay. The results of micronuclei measurements are seen in Fig. 4. Although there is a fair bit of scatter, the results show that there is no difference between the irradiated and unirradiated malathion samples. A linear-regression-fit of the data of Fig. 4 showed that the slope was not significantly different from zero ( p = 0.05) for both samples. We were able to use higher dose levels with the Molt-4 cells than with the V79 cells since malathion was less toxic in the Molt-4 cells as shown in Table 3. The experimental conditions, however, were different for the Molt-4 cells: cell toxicity was determined by the percent of dead cells and the increase in cell number after 3 d of incubation in the presence of malathion, in contrast to colony counting in V79 cells. The main objective of this study was to determine if irradiation effected the toxicity of the organophosphorus pesticide malathion in selected in vitro tests. We concluded that for the end-points measured, a 10 kGy y-exposure did not alter the toxicity significantly. This contrasts with some very early work, which showed a large decrease in insect toxicity of malathion dissolved in hexane after a radiation exposure (Lippold et al., 1969), but our results are qualitatively in
accord with those obtained by Cogburn and Mahany (1969), who irradiated malathion in a dry state without a solvent. Our experiments showed a slight decrease in cell killing and polyploid formation after a radiation exposure; but none of the changes was statistically significant. Also there was a small, nonsignificant increase in SCEs produced by irradiated as compared to unirradiated malathion. This is probably another manifestation of the small decrease in cell killing and polyploidy in the irradiated malathion. More cells completed a second division in the irradiated malathion and could be counted in the SCE analysis. Most polyploid cells are held at the first mitosis and are not analysed for SCE or micronuclei, but they may still carry chromosomal damage. In general, malathion, at the doses we tested, did not cause SCE or micronuclei formation. The principle effect of malathion was polyploid formation and that was probably the cause of cell killing reported in Fig. 1. There is not much information in the literature that describes this aspect of malathion. It was noted, however, in the study by Chen et al. (1981), where they showed 50% of V79 cells were polyploid in the first cell cycle after exposure to 40 > g / m l of malathion. The polyploid levels we report are higher; this may reflect the high-purity malathion samples we used.
TABLE 3 T O X I C I T Y OF M A L A T H I O N ON MOLT-4 CELLS MEAS U R E D A F T E R 3-d E X P O S U R E Each value is the mean of three Expts. Malathion concentration (>g/ml)
D e a d cells (%)
Cell number (10 ~ × c e l l s / m l )
240 120 80 6/) 30 15 7.5 0.0 " 0.0
49 30 12 2.8 3.0 1.6 1.0 1.6 1.1
0.19 0.14 0.36 0.43 0.67 0.75 1.1 0.93 1/.98
a Solvent control.
193 I n c o n c l u s i o n , m a l a t h i o n p r o d u c e d a s m a l l inc r e a s e i n s i s t e r - c h r o m a t i d e x c h a n g e in V 7 9 cells. The main effect of malathion was polyploidy form a t i o n . I r r a d i a t i o n o f m a l a t h i o n d i d n o t a l t e r its t o x i c i t y t o a s i g n i f i c a n t l e v e l in a n y o f t h e t o x i c i t y assays we tested.
References Carcinogen Program NCI (1977) Bioassay of malathion for possible carcinogenicity, Natl. Tech. Inform. Serv., PB-278; 527: 98. Chen, H.H., J.L. Hsueh, S.R. Sirianni and C.C. Huang (1981) Induction of sister-chromatid exchanges and cell cycle delay in cultured mammalian cells treated with eight organophosphorus pesticides, Mutation Res., 88, 307-316. Chen, H.H., S.R. Sirianni and C.C. Huang (1982) Sister chromatid exchanges in Chinese hamster cells treated with seventeen organophosphorus compounds in the presence of a metabolic activation system, Environ. Mutagen., 4, 621-624. Cogburn, R.R., and P.C. Mahany (1969) Effect of gamma radiation on the insecticidal efficiency of malathion deposits on wheat and kraft paper, J. Econ. Entomol., 62, 829-831. Fan, A.M., and J.J. Jackson (1989) Pesticides and food safety, Regul. Toxicol. Pharmacol., 9, 158-174. Frank, R., H.E. Braun and B.D. Ripley (1987) Residues of insecticides, fungicides, and herbicides on Ontario-grown vegetables, 1980-1985, J. Assoc. Anal. Chem., 70, 1081 1086. Frank, R., H.E. Braun and B.D. Ripley (1989) Monitoring Ontario-grown apples for pest control chemicals used in their production, 1978-86, Food Additives and Contaminants, 6, 227-234. Garry, V.F., R.L. Nelson, J. Griffith and M. Harkins (1990) Preparation for human study of pesticide applicators: sister chromatid exchanges and chromosome aberrations in cultured human lymphocytes exposed to selected fumigants, Teratogen. Carcinogen. Mutagen., 10, 21-29. Goto, K., Maeda, Y. Kano and T. Sugiyama (1978) Factors
involved in differential Giemsa-staining of sister chromatids, Chromosoma, 66, 351-359. Hansard (1990) The Parliamentary Debates, House of Lords Official Report No. 1456, Great Britain, pp. 598-560. Hayashi, M., T. Sofuni and M. Ishidate Jr. (1983) An application of acridine orange fluorescent staining to the micronucleus test, Mutation Res., 120, 241-247. Her Majesty's Stationary Office (1986) The safety and wholesomeness of irradiated foods, London, p. 17. Herath, J.F., S.M. Jalal, M.J. Ebertz and J.T. Martsolf (1989) Genotoxicity of the organophosphorus insecticide malathion based on human lymphocytes in culture, Cytologia, 54, 191-195. Huang, C.C. (1973) Effect on growth but not on chromosomes of the mammalian cells after treatment with three organophosphorus insecticides, Proc. Soc. Exp. Biol. Med., 142, 36-40. Lepine, F.L. (1991) Effects of ionizing radiation on pesticides in a food irradiation perspective: A bibliographic review. J. Agric. Food Chem., 38, 2112 2118. Lippold, P.C., J.S. Cleere, L.M. Massey Jr., J.B. Bourke and A.W. Avens (1969) Degradation of insecticides by cobalt-60 gamma radiation, J. Econ. Entomol., 62, 1509-1510. Marquis, J.K. (1989) General toxicology of pesticides in: J.K. Marquid (Ed.), A Guide to General Toxicology, 2nd edn., Karger, Basel, pp. 157-178. Nicholas, A.H., M. Vienne and H. Van Den Berghe (1979) Induction of sister-chromatid exchanges in cultured human cells by an organophosphorus insecticide: malathion, Mutation Res., 67, 167-172. Nishio, A., and E.M. Uyeki (1981) Induction of sister chromatid exchanges in Chinese hamster ovary cells by organophosphate insecticides and their oxygen analogs, J. Toxicol. Environ. Health, 8, 939-946. Shirasu, Y., M. Moriya, K. Kato, A. Furuhashi and T. Kada (1976) Mutagenicity screening of pesticides in the microbial system, Mutation Res., 40, 19-30. The Merck Index 7th edn., S. Budavari (Ed). (1989) Merck, Rahway, N J, p. 5586. Wild, D. (1975) Mutagenicity studies on organophosphorus insecticides, Mutation Res., 32, 133-150. Wong, P.K, C.C. Wai and E. Liong (1989) Comparative study on mutagenicities of organophosphorus insecticides in Salmonella, Chemosphere, 18, 2413-2422.
