FUNDAMENTAL
AND APPLIED
TOXICOLOGY
18,
155-t 59 (1992)
Acute Toxic Effects of Oxamyl in the Rat V. FAYEZ AND W. W. KILGORE Department of Environmental Toxicology, University of CaliJornia, Davis, Cal$ornia 95616
ReceivedApril 23, 1991: acceptedJuly 18, I99 1 When given by intraperitoneal injection, oxamyl is highly Acute Toxic Effects of Oxamyl in Rats. FAYEZ, V., AND toxic to rats, mice and guinea pigs. It is both a mild eye KILGORE,W. W. (1992). Fundam. Appl. Toxicol. l&155-159. irritant, with the reaction limited to the conjunctiva and iris, and a mild skin irritatant, with a dermal LD50 of > 1200 The effects of single acute oral doses of 1, 2.1, and 3.5 mg/ kg oxamyl (a carbamate insecticide) on selected biochemical pa- mg/kg in rats and 740 mg/kg in rabbits (Kennedy, 1986a). rameters in male Sprague-Dawley rats were investigated. The It is also highly toxic both via inhalation, with the 1-hr LC50 animals exhibited significantly decreased weight gain when value in rats being 0.17 mg/liter (male) and 0.12 mg/liter compared to control animals. The compound inhibited brain (female), and when given as a single oral dose, with an LD50 and blood acetycholinesterase significantly in the first few hours of 2.5 to 3.1 mg/kg in fasted rats and 2.3 to 3.3 mg/kg in of exposure. Liver glucose-6-phosphatase was inhibited sub- fasted mice. In all species, oxamyl showed clinical signs of stantially after 7 and 4 days at the levels of 2.1 and 3.5 mg/kg, cholinesterase inhibition. respectively. Maximum inhibition of liver succinic acid dehyIn a chronic toxicity study, rats fed a diet containing oxdrogenase was noted after 1 day at the level of 1 mg/kg and after amyl at 500 ppm for 90 days showed clinical signs of cho6 hr at the level of 2.1 and 3.5 mg/kg. Significant changes in linesterase inhibition and body weight loss within 2 days. A serum total lipids and glucose were observed when oxamyl was feeding period of 2 years also showed depressed body weight given at 2.1 and 3.5 mg/kg, but serum protein was not affected at any dose level. However, the absence of statistically significant gains in rats fed either 100 or 150 ppm. Cholinesterase was effects between Days 7 and 14 in most of the investigated pa- depressed only during the first week of feeding and only in rameters is indicative of an overall moderate degree of toxicity the 150 ppm group (Kennedy, 1986b). of oxamyl following acute oral administration of the selected The present study extends the acute toxicity studies with doses. 0 1992 Society of Toxicology. oxamyl (Kennedy, 1986a) and was undertaken to more completely assess the acute toxicity of the compound and to determine both the rate of recovery and the dose-response Oxamyl, methyl N’,N’-dimethyl-N-[(methylcarbarelationships for various biochemical parameters. moyl)oxy]- 1-thiooxamimidate, is the active ingredient in Vydate insecticidelnematicide and is used widely for conMATERIALS AND METHODS trolling insects, mites, and nematodes (Brown, 1972; Riedel Chemicals. Oxamyl is a white crystalline solid, with a slightly sulfurous et al., 1973; Whitehead et al., 1980; Helyer et al., 1984; and odor, molecular weight of 219.3, specific gravity of 0.97 (25”C/4’C) and Mohammad and Hague, 1987). vapor pressure of 2.3 X 10m4mm Hg (25°C). It is stable in solid form and In plants, its major route of degradation involves the hy- decomposes to innocuous materials in water and in soil. drolysis to the corresponding oximino compound which in Oxamyl used in these studies was produced and supplied by the Biochemicals Department. E. I. du Pont de Nemours and Co., Inc. (Wilmingturn becomes conjugated with glucose. Further metabolism includes demethylation and addition of a glucose unit to the ton, DE). Animals. Male Sprague-Dawley rats (190-2 10 g) were purchased from original conjugate (Harvey et al., 1978). Bantin and Kingman (Fremont, CA) and housed in environmentally conIn rats, oxamyl is degraded by hydrolysis to the oximino trolled rooms (22-24’C; 40-50% relative humidity; 12-hr fluorescent light/ compound or converted enzymatically to N,N-dimethyloxdark cycle) for I week before dosing. Deionized tap water and food (Purina amic acid. Conjugates of these two compounds as well as Rodent Chow) were available ad libitum. Animals were fasted for 12 hr monomethyl derivatives are the major elimination products before dosing and offered the food 2 hr after dosing. in urine and feces (Harvey and Han, 1978). Oxumyl exposure. Oxamyl, with an acute oral LD50 of 5.4 mg/kg to male rats, was dissolved in water at the levels of 1, 2.1, and 3.5 mg/kg of Oxamyl is moderately toxic to aquatic animals. When rat body weight (0.5 ml solution/200 g rat). Dose selection was based on bobwhites and rabbits were oversprayed with the compound using the range of ca. 19, 39, and 65% of the reported LD50, respectively. under field conditions (three times at 3 pounds per acre) no This enabled us to statistically evaluate the difference in the magnitude of clinical or gross pathological effects were produced (Smith, toxicity. Acute oral dosing was done early in the morning by stomach in1982). tubation. For each dose level, assayswere performed on control rats that 155 Copyright All rights
0272-0590/92 $3.00 0 1992 by the Society of Toxicology. of reproduction in any form reserved.
156
FAYEZ AND KILGORE
TABLE 1 Initial Body Weight and Weight Gain in Grams Following Acute Oral Dosesof Oxamyl Body weight gain (day intervals) Oxamyl @x/kg)
Initial body weightW
0.0 1.0 2.1 3.5
202 213 202 192
1 22.1 f 17.0 f 7.5 + 1.3 +
2 3.7” 2.2* 1.7* 4.2*
28.0 20.3 22.0 9.8
+ 2.2 2 3.1* f 1.6’ f 12.3*
3 37.8 k 30.0 f 26.5 + 19.3 +
4 3.3 5.5 4.0* 4.3*
41.3 33.3 44.0 32.3
7
+ iz + +
7.8 4.5 4.2 5.6
64.0 48.5 67.0 61.0
10
r!z6.2 f 3.3* + 8.3 -+ 7.3
82.8 73.3 88.8 79.5
f k + f
14 14.9 3.5 16.1 11.1
104.8 78.5 75.0 78.3
+- 16.2 + 12.8* 2 13.7* rt 10.6*
a Mean + standard deviation of N = 4. * Statistically different from control value, p < 0.05 (Student’s f test).
were handled in a manner equivalent to that of the experimental groups except that dosing of only the water was given. Methods. Brain acetylcholinesterase (AchE) was determined according to a modified Ellman procedure (Gorum et al., 1978). Blood AchE was determined using [ 1-acet~&‘~C]acetylcholine chloride as a substrate (Winteringham and Disney, 1964) in which 20 ~1 of blood is transferred in a heparinized pipet to a tube containing 0.2 ml of hemolyzing solution and mixed. Then 20 ~1 of the diluted blood was placed in the center of a numbered plastic sheet, 12 X 1 in. The reaction was started by adding 10 ~1 of the radioactive substrate (0.000 15 M, - 1500 cpm) and mixing was aided during incubation by gently shaking the plastic sheet by hand. After exactly 2 min, the reaction was stopped by adding 10 ~1 of 1 N HCI and the sheet was immediately dried in hot air using a domestic hair dryer. Then the dry spots containing the residual unhydrolyzed substrate were cut off the plastic sheet, placed in scintillation vials containing 15 ml of scintillation cocktail, and counted by a liquid scintillation spectrometer. Correction for quench was determined using external standardization. Liver SDHase was determined aher the method of Shelton and Rice ( 1957). Liver glucose-6-phosphatase (G6Pase) was determined by the method of Traiger and Plaa (197 I) and the resulting inorganic phosphorus was assayed according to Fiske and Subba Row (1925). Other investigated parameters included the assessment of serum total lipids (Knight et al., 1972) serum total proteins (Gomall et al., 1949). and serum glucose (Somogyi, 1945). Radioactivity was measured using a Packard Tricarb Model 2450 liquid scintillation spectrometer. Optical density was measured using a Varian Gary 2 19 spectrophotometer. Animals were euthanized using slight ether anesthesia at intervals of 3 2nd 6 hr, 1,2,3,4,7, 10, and 14 days after acute dosing. Blood was withdrawn by cardiac puncture and blood cholinesterase was analyzed immediately, after which the plasma was separated and refrigerated for further analysis. Further, more animals were euthanized at shorter intervals of f, 1 and 2 hr only for the measurement of brain and blood acetylcholinesterase to determine the time at which oxamyl manifests its maximum toxic effect on those
two enzymes. Animals were quickly dissected after blood collection. Brain and liver tissues were rinsed with ice-cold isotonic saline, blotted dry, and analyzed on the same day for brain AchE, liver succinic acid dehydrogenase (SDHase), and G6Pase. Statistical analysis. Student’s t test was employed and the level of significance was set at p c 0.05.
RESULTS
Growth rate. The mean body weigbt gains for each group recorded at Days 1, 2, 3,4, 7, 10, and 14 are given in Table 1. The weight gains of the animals were significantly lower during the first 3 days in the 2. I and 3.5 mg/kg groups. The reduced weight gain in the 1 mg/kg group was significant in the first 2 days and after 7 days. However, the three dose levels showed significant reduction in body weight after 14 days. Clinical observations. Following dosing, the animals were continuously observed for the first 6 hr and daily for 14 days to determine any clinical symptoms or changes in gross behavior. Signs of cholinesterase inhibition were seen almost 20 min after treatment and persisted for about 5 hr depending on the dose level. It included salivation, occular changes, tremors, fasciculations, and lacrimation. One rat treated with 3.5 mg/kg died after 90 min. Brain acetylcholinesterase. Table 2 shows the percentage inhibition of the enzyme activity. Maximum inhibition to 46% of control levels was noted at 2 hr after dosing with 3.5
TABLE 2 Activity of Brain Acetylcholinesterase,Expressedas PercentageInhibition, Following Acute Oral Dosesof Oxamyl Time
DO% 1 w/kg 2.1 mg/kg 3.5 mg/kg
0.5 25 f 3.v 36 + 6.2’ 33 k 12.5;
I 40 * 5.5’ 42 f 5.9’ 31 * 3.4’
in
hours
2 15-+9.v 16.7 f 19.5 46 2 8.9’
Time 3 17.5 t 9.2* 39 * 6.8’
6 19 f 8.3*
a Mean f standard deviation of N = 4. * Significantly different from control value, p < 0.05 (Student’s t test).
in days
1
2
3
4
3.4 2 1.6 8 + 15.0 6.8 k 6.2
5.3 f 1.9 1.1+2.1 6 + 9.5
5.5 t 0.8 0.5 + 1.0
1.4 f 0.8 4.3 * 1.4
7
6.5 k 8.5
10
14
1.9 f 1.6 -
0.8 + 1.5 3.3 + 5.2
EFFECTS OF OXAMYL
TABLE 3 Activity of Blood Acetylcholinesterase, Expressed as Percentage Inhibition, Time Dose
0.5
1 mg/kg 2.1 m&kg 3.5 mg/kg
’ Mean + standard deviation * Significantly different from
Time
2
75 k 12.9* 72 + 4.9* 71 t 13.0’
Following Acute Oral Doses of Oxamyl
in hours
I
68 ? 8.5”* 83 k 4.5* 74 t 4.9*
157
IN RAT
of N = 4. control value,
6
1
2
3
4
I
15 3~ 11.6 9 + 9.1 6.3 + 10.0
1.4 It 2.7 7.5 2 3.7 12 i 9.3
3.5 + 2.8 8 it 7.9
11.5 ? 6.4 19 + 1.4
1.5 + 1.9 16 f 6.6
2 2 4.0 I i 2.0 427.1
3
46 ‘- 11.0’ 63 f 17.0* 82 f 3.5*
10 + 6.7 37 f 4.6* 64 t 5.9*
p -C 0.05
in days IO 7 * 5.3 3.8 + 3.5 8 2 0.95
14 4.3 + 5.9 10 k 8.5 -
t test).
(Student’s
mg/kg and the level remained depressed for 6 hr. The groups of animals dosed with 1 mg/kg and 2.1 mg/kg showed maximum inhibition of the enzyme activity after 1 hr, with the effect lasting for 2 and 3 hr, respectively, after dosing. Blood aceylcholinesterase. Table 3 illustrates the inhibition of blood AchE. The enzyme activity was significantly inhibited during the first 2 hr at the level of 1 mg/kg and during the first 3 hr at the level of 2.1 and 3.5 mg/kg. The enzyme activity was not significantly decreased any further at 6 hr and thereafter. Liver glucose6phosphatase and succinic acid dehydrogenase. The response of the liver to oxamyl was estimated
through the measurement of G6Pase and SDHase. Table 4 shows the activity of G6Pase expressed in mg Pi/g liver/20 min. A slight, significant reduction in the enzyme activity could be observed after 7 and 4 days at the level of 2.1 and 3.5 mgfkg, respectively. However, the enzyme activity had returned to normal after 14 days. The group of animals dosed with 1 mg/kg did not show any depression of the enzyme activity. Succinic dehydrogenase activity is shown in Table 5. Maximum inhibition of the enzyme activity was observed after 1 day and 6 hr at the levels of 1 and 2.1 mg/kg respectively with no dose-response relationship. The animals dosed with 3.5 mg/kg suffered maximum inhibition of the enzyme activity after 1 day and it remained significantly depressed for 2 days. At all dose levels the enzyme activity had returned to normal after 2 days.
Changes in some blood constituents. Table 6 shows the changes in serum total lipids, glucose and total protein compared to the control. A significant increase in total lipids compared to the control was observed only after 3 hr at the levels of 2.1 and 3.5 w/k. At the level of 1 mg/kg oxamyl had no effect on blood glucose at any time intervals, but it caused significant hyperglycemia after 3 hr at the level of 2.1 mg/kg. The highest dose level (3.5 mg/kg) resulted in hyperglycemia at the 3 2nd 6-hr and l-day intervals. On the other hand, the same dose level led to hypoglycemia between the 4th and the 10th day. Concerning the total protein level in serum, it can be seen that there is no apparent significant difference between the control and the test groups at any time intervals.
DISCUSSION The data obtained in the present investigation demonstrate that acute poisoning of rats by different doses of oxamyl resulted in great inhibition of brain aceylcholinesterase. Although the extent of inhibition was not dose-related in the first hour, it can be stated that the magnitude of inhibition progressed more severely and lasted longer as the dose level increased. However, this inhibition did not persist for more than 6 hr due to the unstable carbamylated AchE which leads to the regeneration of the enzyme. On the other hand, blood acetylcholinesterase was much more affected by the insecticide compared to the brain acetylcholinesterase, and
TABLE 4 Activity of Liver Glucose-6-Phosphatase, Expressed as mg Pi/g Liver/20 min, following Acute Oral Dosesof Oxamy Time Dose
0.5
1
0 w/kg a/kg 2.11 mg/kg 3.5 mg.lkg
2
in hours
Time
3 6.13 5.65 6.1 5.98
+ ?I+ iz
6 0.57” 0.37 0.32 0.29
6.1 5.53 5.15 7.02
+ +f f
1 0.36 0.88 0.31 1.50
6.4 6.25 6.8 7.38
2 f f it
2 0.29 1.40 0.49 0.90
5.9 5.3 1.08 5.35
r?Mean rt standard deviation of N = 4. * Significantly
different
from
control
value,
p < 0.05
(Student’s
t test).
* f f +
3 0.22 0.89 1.10 1.10
5.68 5.25 4.45 5.53
+ f * k
in days 4
0.45 0.89 1.10 0.24
6.2 6.95 6.75 4.75
f f f k
I 0.47 0.94 0.5 I 0.54’
5.98 5.9 4.45 5.7
+ + + f
10 0.51 0.35 0.84* 1.10
6.13 6.02 6.35 5.55
+ f + f
14 0.34 0.25 0.52 0.13*
5.8 5.7 6.3 5.28
+ k f +
0.21 0.80 0.70 0.45
158
FAYEZ
AND KILGORE
TABLE 5 Activity of Liver Succinic Dehydrogenase, Expressed as Percentage Inhibition, Time Dose
0.5
1
2
1 m/kg 2.1 mg/kg 3.5 me/ka
Following Acute Oral Doses of Oxamyl
in hours
Time
3
6
21 * 21.3 9.4 + 10.7 3.9 + 7.8
3.4 + 4.5 21.4 31 9.2* 17 k 10.8*
1 24 iz 2.9* 1.6 f 3.2 28.8 f 18.3
in days
2
3
4
7
10
14
6.2 * 5.3 2.3 3~ 4.6 13.7 k 8.2
3.8 ic 4.0 6.4 + 6.3 -
4.3 f 4.9 5.2 + 4.8
1.6 f 1.9 8.6 k 1.6 9.5 k 8.4
1.4 k 2.2 11.3 * 4.1 -
5.9 * 4.3 4.9 ? 9.7 5.3 k 1.4
n Mean + standard deviation of N = 4. * Significantly different from control value. p -C 0.05 (Student’s t test).
a maximum inhibition of 82% was reached after 2 hr at 3.5 mg/kg. However, recovery of both enzymes was almost parallel. Body weight gain was reduced significantly almost over the entire first 3 days after dosing. This is related to a reduced food consumption of the treated animals compared to the corresponding controls. On the other hand, the reason for the significantly reduced weight gain observed after 14 days at the three dose levels is not clear. Concerning mortality, only 1 animal out of 48 dosed with 3.5 mg/kg oxamyl died after 1.5 hr. In this respect, Kennedy (1986a) reported a much higher incidence of mortality in male albino rats dosed with acute doses ranging from 2-5 mglk. The hyperglycemia observed in the early period after dosing at the level of 2.1 and 3.5 mg/kg nearly coincided with the onset and severity of tremors and persisted longer at the highest dose level (Table 2). This might be explained by the
fact that the inhibition of cholinesterase leads to the hypersecretion of epinephrine, which stimulates the breakdown of residual liver glycogen to glucose (Murphy and Porter, 1966). In this connection, it is worth mentioning that earlier investigators reported insecticide-induced hyperglycemia after acute poisoning of rats by the organophosphate insecticides delnav, dipterex, and malathion and the organochlorine compound dieldrin (Murphy, 1966). However, oxamyl resulted in significant hypoglycemia when given at 3.5 mg/kg between the 7th and 10th day, which might be attributed to a certain mechanism that induces the secretion of insulin. Liver G6Pase and SDHase were determined to evaluate the effect of oxamyl on the integrity of the hepatic endoplasmic reticulum and the mitochondria, respectively. Table 3 (G6Pase) and Table 4 (SDHase) illustrate that, although the activity of the two enzymes was significantly inhibited at certain intervals, it returned to normal thereafter. This observation is suggestive of the recovery of the target organ.
TABLE 6 Effect of Acute Oral Doses of Oxamyl on Some Blood Constituents Time Dose bWkg)
3 hr
6 hr
1 day
2 days Total
0 1 2.1 3.5
171 189 238 332
f 16.5” ? 27.8 IT 19.6’ *49.1*
231 237 234 282
+40.1 zk 63.5 f 44.6 k 24.1
242 253 278 231
31 k 2 f
0 1 2.1 3.5
83.4 81.1 114.5 113.9
k 4.7 T 4.9 k 19.1’ + 6.1’
82.6 96.1 80.9 108.3
f f zk k
83.4 86.8 82 102.3
+ 7.2 k 15.3 k 5.4 k 7.1*
0 I 2.1 3.5
6.1 5.6 6.4 6.8
6.6 6.1 6.3 7.1
+ f ? f
26.1 44.9 19.1 19.4
279 255 218 219
lipids
0.53 0.2 0.25 0.33
.08 0.29 0.91 0.81
6.3 6.3 6.5 6
+ f + zk
0.6 0.2 0.14 0.9
6.3 6.3 6.4 5.8
280 261 251 298
f f + f
protein 0.13 0.08 0.14 0.74
’ Mean f standard deviation of N = 4. * Significantly different from control value, p < 0.05 (Student’s f test).
4 days
7 days
10 days
14 days
ml serum) + 11.9 f29 +24 1? 40.7
(mgjl00
& 13 f 1.5 k 10 + 37.1
Total rt f zt +
(mg/lOO
+ 18.9 + 9.1 * 15.1 + 30.4
89.4 83.2 87.3 110.2
after dosing
3 days
Glucose 1 9.2 16.7 9.2*
intervals
286 292 259 371
f 2.9 + 25.8 + 39.5 + 182
258 252 291 293
+ + + k
27.8 43.8 16.7 46.3
286 285 291 377
+ zt + k
42.5 51.4 16.7 11.6
263 279 304 297
Z!I 18.4 + 17.1 ? 31.2 + 15.2
88.3 75.3 94.2 101.1
+ 8.6 i: 7.1 + 10.6 + 14.6
ml serum) 95.1 82.1 88 92.9
(g/100 6.5 6.5 6.7 6
f 21.6 + 8.3 k 13.5 + 19.7
92.2 77.5 89.5 78.6
+ 2 + k
6.6 6.7 6.5 5.6
k f + ct
15.5 4.9 7.4 5
98.8 86.3 87.3 81.5
+ 11.4 & 2.8 + 8.5 A 5.2*
86.5 76.5 99.9 13
* 7.3 + 12.1 2 10.1 rt 3.1’
ml serum) f + f +
0.19 0.21 0.06 0.9
0.4 0.09 0.32 1.2
6.4 6.5 6.4 7.2
+ k + +
0.25 0.06 0.2 0.79
7.6 8.1 I 7.9
+ + + k
0.5 0.09 0.4 0.6
7 1.2 7.1 7.1
+ 2 z? *
0.2 0.2 0.2 0.4
EFFECTS OF OXAMYL
In this respect, it should be stated that earlier investigators reported alterations of liver glucose-6-phosphatase (Kuz’uminskaya and Yakushko, 1970; Panek et al., 1973) and succinic dehydrogenase (Wojcik et al., 1974; Sitkiewicz and Zalewska, 1975; Carlson and Dubois, 1970) in rats and mice after exposure to certain pesticides. Concerning the total lipids in serum (Table 2), the elevation observed at the level of 2.1 and 3.5 mg/kg after 3 hr might be attributed to inducing the mobilization of the fatty acids from the adipose tissue triglyceride. On the other hand, it can be concluded that the insecticide did not affect protein metabolism at any dose level, so that the rate of protein degradation was balanced with the rate of synthesis. Moreover, absence of depressed serum protein levels means that oxamyl did not affect liver parenchymal cells responsible for synthesis of most serum proteins. Generally, in terms of what might be anticipated from such a compound with a low LD50, the data suggest that the maximum response of the studied parameters had not been reached, although higher doses are within the lethal range of oxamyl. Apparently it derives its toxic effect from being a potent anticholinesterase agent, but in general its acute toxicity to rats is of moderate order. In summary, the data reported herein is suggestive of a reversible toxic effect of oxamyl on the investigated parameters. These results suggest that further work, including histopathological examination as well as clinical tests, is necessary to elucidate the possibility of irreversible damage following oxamyl exposure. ACKNOWLEDGMENTS The authors acknowledge and thank Jerry Ito for statistical analysis and manuscript preparation.
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Zakl.
Hig. 25(6),
6 19-626.
AND APPLIED
TOXICOLOGY
18,
155-t 59 (1992)
Acute Toxic Effects of Oxamyl in the Rat V. FAYEZ AND W. W. KILGORE Department of Environmental Toxicology, University of CaliJornia, Davis, Cal$ornia 95616
ReceivedApril 23, 1991: acceptedJuly 18, I99 1 When given by intraperitoneal injection, oxamyl is highly Acute Toxic Effects of Oxamyl in Rats. FAYEZ, V., AND toxic to rats, mice and guinea pigs. It is both a mild eye KILGORE,W. W. (1992). Fundam. Appl. Toxicol. l&155-159. irritant, with the reaction limited to the conjunctiva and iris, and a mild skin irritatant, with a dermal LD50 of > 1200 The effects of single acute oral doses of 1, 2.1, and 3.5 mg/ kg oxamyl (a carbamate insecticide) on selected biochemical pa- mg/kg in rats and 740 mg/kg in rabbits (Kennedy, 1986a). rameters in male Sprague-Dawley rats were investigated. The It is also highly toxic both via inhalation, with the 1-hr LC50 animals exhibited significantly decreased weight gain when value in rats being 0.17 mg/liter (male) and 0.12 mg/liter compared to control animals. The compound inhibited brain (female), and when given as a single oral dose, with an LD50 and blood acetycholinesterase significantly in the first few hours of 2.5 to 3.1 mg/kg in fasted rats and 2.3 to 3.3 mg/kg in of exposure. Liver glucose-6-phosphatase was inhibited sub- fasted mice. In all species, oxamyl showed clinical signs of stantially after 7 and 4 days at the levels of 2.1 and 3.5 mg/kg, cholinesterase inhibition. respectively. Maximum inhibition of liver succinic acid dehyIn a chronic toxicity study, rats fed a diet containing oxdrogenase was noted after 1 day at the level of 1 mg/kg and after amyl at 500 ppm for 90 days showed clinical signs of cho6 hr at the level of 2.1 and 3.5 mg/kg. Significant changes in linesterase inhibition and body weight loss within 2 days. A serum total lipids and glucose were observed when oxamyl was feeding period of 2 years also showed depressed body weight given at 2.1 and 3.5 mg/kg, but serum protein was not affected at any dose level. However, the absence of statistically significant gains in rats fed either 100 or 150 ppm. Cholinesterase was effects between Days 7 and 14 in most of the investigated pa- depressed only during the first week of feeding and only in rameters is indicative of an overall moderate degree of toxicity the 150 ppm group (Kennedy, 1986b). of oxamyl following acute oral administration of the selected The present study extends the acute toxicity studies with doses. 0 1992 Society of Toxicology. oxamyl (Kennedy, 1986a) and was undertaken to more completely assess the acute toxicity of the compound and to determine both the rate of recovery and the dose-response Oxamyl, methyl N’,N’-dimethyl-N-[(methylcarbarelationships for various biochemical parameters. moyl)oxy]- 1-thiooxamimidate, is the active ingredient in Vydate insecticidelnematicide and is used widely for conMATERIALS AND METHODS trolling insects, mites, and nematodes (Brown, 1972; Riedel Chemicals. Oxamyl is a white crystalline solid, with a slightly sulfurous et al., 1973; Whitehead et al., 1980; Helyer et al., 1984; and odor, molecular weight of 219.3, specific gravity of 0.97 (25”C/4’C) and Mohammad and Hague, 1987). vapor pressure of 2.3 X 10m4mm Hg (25°C). It is stable in solid form and In plants, its major route of degradation involves the hy- decomposes to innocuous materials in water and in soil. drolysis to the corresponding oximino compound which in Oxamyl used in these studies was produced and supplied by the Biochemicals Department. E. I. du Pont de Nemours and Co., Inc. (Wilmingturn becomes conjugated with glucose. Further metabolism includes demethylation and addition of a glucose unit to the ton, DE). Animals. Male Sprague-Dawley rats (190-2 10 g) were purchased from original conjugate (Harvey et al., 1978). Bantin and Kingman (Fremont, CA) and housed in environmentally conIn rats, oxamyl is degraded by hydrolysis to the oximino trolled rooms (22-24’C; 40-50% relative humidity; 12-hr fluorescent light/ compound or converted enzymatically to N,N-dimethyloxdark cycle) for I week before dosing. Deionized tap water and food (Purina amic acid. Conjugates of these two compounds as well as Rodent Chow) were available ad libitum. Animals were fasted for 12 hr monomethyl derivatives are the major elimination products before dosing and offered the food 2 hr after dosing. in urine and feces (Harvey and Han, 1978). Oxumyl exposure. Oxamyl, with an acute oral LD50 of 5.4 mg/kg to male rats, was dissolved in water at the levels of 1, 2.1, and 3.5 mg/kg of Oxamyl is moderately toxic to aquatic animals. When rat body weight (0.5 ml solution/200 g rat). Dose selection was based on bobwhites and rabbits were oversprayed with the compound using the range of ca. 19, 39, and 65% of the reported LD50, respectively. under field conditions (three times at 3 pounds per acre) no This enabled us to statistically evaluate the difference in the magnitude of clinical or gross pathological effects were produced (Smith, toxicity. Acute oral dosing was done early in the morning by stomach in1982). tubation. For each dose level, assayswere performed on control rats that 155 Copyright All rights
0272-0590/92 $3.00 0 1992 by the Society of Toxicology. of reproduction in any form reserved.
156
FAYEZ AND KILGORE
TABLE 1 Initial Body Weight and Weight Gain in Grams Following Acute Oral Dosesof Oxamyl Body weight gain (day intervals) Oxamyl @x/kg)
Initial body weightW
0.0 1.0 2.1 3.5
202 213 202 192
1 22.1 f 17.0 f 7.5 + 1.3 +
2 3.7” 2.2* 1.7* 4.2*
28.0 20.3 22.0 9.8
+ 2.2 2 3.1* f 1.6’ f 12.3*
3 37.8 k 30.0 f 26.5 + 19.3 +
4 3.3 5.5 4.0* 4.3*
41.3 33.3 44.0 32.3
7
+ iz + +
7.8 4.5 4.2 5.6
64.0 48.5 67.0 61.0
10
r!z6.2 f 3.3* + 8.3 -+ 7.3
82.8 73.3 88.8 79.5
f k + f
14 14.9 3.5 16.1 11.1
104.8 78.5 75.0 78.3
+- 16.2 + 12.8* 2 13.7* rt 10.6*
a Mean + standard deviation of N = 4. * Statistically different from control value, p < 0.05 (Student’s f test).
were handled in a manner equivalent to that of the experimental groups except that dosing of only the water was given. Methods. Brain acetylcholinesterase (AchE) was determined according to a modified Ellman procedure (Gorum et al., 1978). Blood AchE was determined using [ 1-acet~&‘~C]acetylcholine chloride as a substrate (Winteringham and Disney, 1964) in which 20 ~1 of blood is transferred in a heparinized pipet to a tube containing 0.2 ml of hemolyzing solution and mixed. Then 20 ~1 of the diluted blood was placed in the center of a numbered plastic sheet, 12 X 1 in. The reaction was started by adding 10 ~1 of the radioactive substrate (0.000 15 M, - 1500 cpm) and mixing was aided during incubation by gently shaking the plastic sheet by hand. After exactly 2 min, the reaction was stopped by adding 10 ~1 of 1 N HCI and the sheet was immediately dried in hot air using a domestic hair dryer. Then the dry spots containing the residual unhydrolyzed substrate were cut off the plastic sheet, placed in scintillation vials containing 15 ml of scintillation cocktail, and counted by a liquid scintillation spectrometer. Correction for quench was determined using external standardization. Liver SDHase was determined aher the method of Shelton and Rice ( 1957). Liver glucose-6-phosphatase (G6Pase) was determined by the method of Traiger and Plaa (197 I) and the resulting inorganic phosphorus was assayed according to Fiske and Subba Row (1925). Other investigated parameters included the assessment of serum total lipids (Knight et al., 1972) serum total proteins (Gomall et al., 1949). and serum glucose (Somogyi, 1945). Radioactivity was measured using a Packard Tricarb Model 2450 liquid scintillation spectrometer. Optical density was measured using a Varian Gary 2 19 spectrophotometer. Animals were euthanized using slight ether anesthesia at intervals of 3 2nd 6 hr, 1,2,3,4,7, 10, and 14 days after acute dosing. Blood was withdrawn by cardiac puncture and blood cholinesterase was analyzed immediately, after which the plasma was separated and refrigerated for further analysis. Further, more animals were euthanized at shorter intervals of f, 1 and 2 hr only for the measurement of brain and blood acetylcholinesterase to determine the time at which oxamyl manifests its maximum toxic effect on those
two enzymes. Animals were quickly dissected after blood collection. Brain and liver tissues were rinsed with ice-cold isotonic saline, blotted dry, and analyzed on the same day for brain AchE, liver succinic acid dehydrogenase (SDHase), and G6Pase. Statistical analysis. Student’s t test was employed and the level of significance was set at p c 0.05.
RESULTS
Growth rate. The mean body weigbt gains for each group recorded at Days 1, 2, 3,4, 7, 10, and 14 are given in Table 1. The weight gains of the animals were significantly lower during the first 3 days in the 2. I and 3.5 mg/kg groups. The reduced weight gain in the 1 mg/kg group was significant in the first 2 days and after 7 days. However, the three dose levels showed significant reduction in body weight after 14 days. Clinical observations. Following dosing, the animals were continuously observed for the first 6 hr and daily for 14 days to determine any clinical symptoms or changes in gross behavior. Signs of cholinesterase inhibition were seen almost 20 min after treatment and persisted for about 5 hr depending on the dose level. It included salivation, occular changes, tremors, fasciculations, and lacrimation. One rat treated with 3.5 mg/kg died after 90 min. Brain acetylcholinesterase. Table 2 shows the percentage inhibition of the enzyme activity. Maximum inhibition to 46% of control levels was noted at 2 hr after dosing with 3.5
TABLE 2 Activity of Brain Acetylcholinesterase,Expressedas PercentageInhibition, Following Acute Oral Dosesof Oxamyl Time
DO% 1 w/kg 2.1 mg/kg 3.5 mg/kg
0.5 25 f 3.v 36 + 6.2’ 33 k 12.5;
I 40 * 5.5’ 42 f 5.9’ 31 * 3.4’
in
hours
2 15-+9.v 16.7 f 19.5 46 2 8.9’
Time 3 17.5 t 9.2* 39 * 6.8’
6 19 f 8.3*
a Mean f standard deviation of N = 4. * Significantly different from control value, p < 0.05 (Student’s t test).
in days
1
2
3
4
3.4 2 1.6 8 + 15.0 6.8 k 6.2
5.3 f 1.9 1.1+2.1 6 + 9.5
5.5 t 0.8 0.5 + 1.0
1.4 f 0.8 4.3 * 1.4
7
6.5 k 8.5
10
14
1.9 f 1.6 -
0.8 + 1.5 3.3 + 5.2
EFFECTS OF OXAMYL
TABLE 3 Activity of Blood Acetylcholinesterase, Expressed as Percentage Inhibition, Time Dose
0.5
1 mg/kg 2.1 m&kg 3.5 mg/kg
’ Mean + standard deviation * Significantly different from
Time
2
75 k 12.9* 72 + 4.9* 71 t 13.0’
Following Acute Oral Doses of Oxamyl
in hours
I
68 ? 8.5”* 83 k 4.5* 74 t 4.9*
157
IN RAT
of N = 4. control value,
6
1
2
3
4
I
15 3~ 11.6 9 + 9.1 6.3 + 10.0
1.4 It 2.7 7.5 2 3.7 12 i 9.3
3.5 + 2.8 8 it 7.9
11.5 ? 6.4 19 + 1.4
1.5 + 1.9 16 f 6.6
2 2 4.0 I i 2.0 427.1
3
46 ‘- 11.0’ 63 f 17.0* 82 f 3.5*
10 + 6.7 37 f 4.6* 64 t 5.9*
p -C 0.05
in days IO 7 * 5.3 3.8 + 3.5 8 2 0.95
14 4.3 + 5.9 10 k 8.5 -
t test).
(Student’s
mg/kg and the level remained depressed for 6 hr. The groups of animals dosed with 1 mg/kg and 2.1 mg/kg showed maximum inhibition of the enzyme activity after 1 hr, with the effect lasting for 2 and 3 hr, respectively, after dosing. Blood aceylcholinesterase. Table 3 illustrates the inhibition of blood AchE. The enzyme activity was significantly inhibited during the first 2 hr at the level of 1 mg/kg and during the first 3 hr at the level of 2.1 and 3.5 mg/kg. The enzyme activity was not significantly decreased any further at 6 hr and thereafter. Liver glucose6phosphatase and succinic acid dehydrogenase. The response of the liver to oxamyl was estimated
through the measurement of G6Pase and SDHase. Table 4 shows the activity of G6Pase expressed in mg Pi/g liver/20 min. A slight, significant reduction in the enzyme activity could be observed after 7 and 4 days at the level of 2.1 and 3.5 mgfkg, respectively. However, the enzyme activity had returned to normal after 14 days. The group of animals dosed with 1 mg/kg did not show any depression of the enzyme activity. Succinic dehydrogenase activity is shown in Table 5. Maximum inhibition of the enzyme activity was observed after 1 day and 6 hr at the levels of 1 and 2.1 mg/kg respectively with no dose-response relationship. The animals dosed with 3.5 mg/kg suffered maximum inhibition of the enzyme activity after 1 day and it remained significantly depressed for 2 days. At all dose levels the enzyme activity had returned to normal after 2 days.
Changes in some blood constituents. Table 6 shows the changes in serum total lipids, glucose and total protein compared to the control. A significant increase in total lipids compared to the control was observed only after 3 hr at the levels of 2.1 and 3.5 w/k. At the level of 1 mg/kg oxamyl had no effect on blood glucose at any time intervals, but it caused significant hyperglycemia after 3 hr at the level of 2.1 mg/kg. The highest dose level (3.5 mg/kg) resulted in hyperglycemia at the 3 2nd 6-hr and l-day intervals. On the other hand, the same dose level led to hypoglycemia between the 4th and the 10th day. Concerning the total protein level in serum, it can be seen that there is no apparent significant difference between the control and the test groups at any time intervals.
DISCUSSION The data obtained in the present investigation demonstrate that acute poisoning of rats by different doses of oxamyl resulted in great inhibition of brain aceylcholinesterase. Although the extent of inhibition was not dose-related in the first hour, it can be stated that the magnitude of inhibition progressed more severely and lasted longer as the dose level increased. However, this inhibition did not persist for more than 6 hr due to the unstable carbamylated AchE which leads to the regeneration of the enzyme. On the other hand, blood acetylcholinesterase was much more affected by the insecticide compared to the brain acetylcholinesterase, and
TABLE 4 Activity of Liver Glucose-6-Phosphatase, Expressed as mg Pi/g Liver/20 min, following Acute Oral Dosesof Oxamy Time Dose
0.5
1
0 w/kg a/kg 2.11 mg/kg 3.5 mg.lkg
2
in hours
Time
3 6.13 5.65 6.1 5.98
+ ?I+ iz
6 0.57” 0.37 0.32 0.29
6.1 5.53 5.15 7.02
+ +f f
1 0.36 0.88 0.31 1.50
6.4 6.25 6.8 7.38
2 f f it
2 0.29 1.40 0.49 0.90
5.9 5.3 1.08 5.35
r?Mean rt standard deviation of N = 4. * Significantly
different
from
control
value,
p < 0.05
(Student’s
t test).
* f f +
3 0.22 0.89 1.10 1.10
5.68 5.25 4.45 5.53
+ f * k
in days 4
0.45 0.89 1.10 0.24
6.2 6.95 6.75 4.75
f f f k
I 0.47 0.94 0.5 I 0.54’
5.98 5.9 4.45 5.7
+ + + f
10 0.51 0.35 0.84* 1.10
6.13 6.02 6.35 5.55
+ f + f
14 0.34 0.25 0.52 0.13*
5.8 5.7 6.3 5.28
+ k f +
0.21 0.80 0.70 0.45
158
FAYEZ
AND KILGORE
TABLE 5 Activity of Liver Succinic Dehydrogenase, Expressed as Percentage Inhibition, Time Dose
0.5
1
2
1 m/kg 2.1 mg/kg 3.5 me/ka
Following Acute Oral Doses of Oxamyl
in hours
Time
3
6
21 * 21.3 9.4 + 10.7 3.9 + 7.8
3.4 + 4.5 21.4 31 9.2* 17 k 10.8*
1 24 iz 2.9* 1.6 f 3.2 28.8 f 18.3
in days
2
3
4
7
10
14
6.2 * 5.3 2.3 3~ 4.6 13.7 k 8.2
3.8 ic 4.0 6.4 + 6.3 -
4.3 f 4.9 5.2 + 4.8
1.6 f 1.9 8.6 k 1.6 9.5 k 8.4
1.4 k 2.2 11.3 * 4.1 -
5.9 * 4.3 4.9 ? 9.7 5.3 k 1.4
n Mean + standard deviation of N = 4. * Significantly different from control value. p -C 0.05 (Student’s t test).
a maximum inhibition of 82% was reached after 2 hr at 3.5 mg/kg. However, recovery of both enzymes was almost parallel. Body weight gain was reduced significantly almost over the entire first 3 days after dosing. This is related to a reduced food consumption of the treated animals compared to the corresponding controls. On the other hand, the reason for the significantly reduced weight gain observed after 14 days at the three dose levels is not clear. Concerning mortality, only 1 animal out of 48 dosed with 3.5 mg/kg oxamyl died after 1.5 hr. In this respect, Kennedy (1986a) reported a much higher incidence of mortality in male albino rats dosed with acute doses ranging from 2-5 mglk. The hyperglycemia observed in the early period after dosing at the level of 2.1 and 3.5 mg/kg nearly coincided with the onset and severity of tremors and persisted longer at the highest dose level (Table 2). This might be explained by the
fact that the inhibition of cholinesterase leads to the hypersecretion of epinephrine, which stimulates the breakdown of residual liver glycogen to glucose (Murphy and Porter, 1966). In this connection, it is worth mentioning that earlier investigators reported insecticide-induced hyperglycemia after acute poisoning of rats by the organophosphate insecticides delnav, dipterex, and malathion and the organochlorine compound dieldrin (Murphy, 1966). However, oxamyl resulted in significant hypoglycemia when given at 3.5 mg/kg between the 7th and 10th day, which might be attributed to a certain mechanism that induces the secretion of insulin. Liver G6Pase and SDHase were determined to evaluate the effect of oxamyl on the integrity of the hepatic endoplasmic reticulum and the mitochondria, respectively. Table 3 (G6Pase) and Table 4 (SDHase) illustrate that, although the activity of the two enzymes was significantly inhibited at certain intervals, it returned to normal thereafter. This observation is suggestive of the recovery of the target organ.
TABLE 6 Effect of Acute Oral Doses of Oxamyl on Some Blood Constituents Time Dose bWkg)
3 hr
6 hr
1 day
2 days Total
0 1 2.1 3.5
171 189 238 332
f 16.5” ? 27.8 IT 19.6’ *49.1*
231 237 234 282
+40.1 zk 63.5 f 44.6 k 24.1
242 253 278 231
31 k 2 f
0 1 2.1 3.5
83.4 81.1 114.5 113.9
k 4.7 T 4.9 k 19.1’ + 6.1’
82.6 96.1 80.9 108.3
f f zk k
83.4 86.8 82 102.3
+ 7.2 k 15.3 k 5.4 k 7.1*
0 I 2.1 3.5
6.1 5.6 6.4 6.8
6.6 6.1 6.3 7.1
+ f ? f
26.1 44.9 19.1 19.4
279 255 218 219
lipids
0.53 0.2 0.25 0.33
.08 0.29 0.91 0.81
6.3 6.3 6.5 6
+ f + zk
0.6 0.2 0.14 0.9
6.3 6.3 6.4 5.8
280 261 251 298
f f + f
protein 0.13 0.08 0.14 0.74
’ Mean f standard deviation of N = 4. * Significantly different from control value, p < 0.05 (Student’s f test).
4 days
7 days
10 days
14 days
ml serum) + 11.9 f29 +24 1? 40.7
(mgjl00
& 13 f 1.5 k 10 + 37.1
Total rt f zt +
(mg/lOO
+ 18.9 + 9.1 * 15.1 + 30.4
89.4 83.2 87.3 110.2
after dosing
3 days
Glucose 1 9.2 16.7 9.2*
intervals
286 292 259 371
f 2.9 + 25.8 + 39.5 + 182
258 252 291 293
+ + + k
27.8 43.8 16.7 46.3
286 285 291 377
+ zt + k
42.5 51.4 16.7 11.6
263 279 304 297
Z!I 18.4 + 17.1 ? 31.2 + 15.2
88.3 75.3 94.2 101.1
+ 8.6 i: 7.1 + 10.6 + 14.6
ml serum) 95.1 82.1 88 92.9
(g/100 6.5 6.5 6.7 6
f 21.6 + 8.3 k 13.5 + 19.7
92.2 77.5 89.5 78.6
+ 2 + k
6.6 6.7 6.5 5.6
k f + ct
15.5 4.9 7.4 5
98.8 86.3 87.3 81.5
+ 11.4 & 2.8 + 8.5 A 5.2*
86.5 76.5 99.9 13
* 7.3 + 12.1 2 10.1 rt 3.1’
ml serum) f + f +
0.19 0.21 0.06 0.9
0.4 0.09 0.32 1.2
6.4 6.5 6.4 7.2
+ k + +
0.25 0.06 0.2 0.79
7.6 8.1 I 7.9
+ + + k
0.5 0.09 0.4 0.6
7 1.2 7.1 7.1
+ 2 z? *
0.2 0.2 0.2 0.4
EFFECTS OF OXAMYL
In this respect, it should be stated that earlier investigators reported alterations of liver glucose-6-phosphatase (Kuz’uminskaya and Yakushko, 1970; Panek et al., 1973) and succinic dehydrogenase (Wojcik et al., 1974; Sitkiewicz and Zalewska, 1975; Carlson and Dubois, 1970) in rats and mice after exposure to certain pesticides. Concerning the total lipids in serum (Table 2), the elevation observed at the level of 2.1 and 3.5 mg/kg after 3 hr might be attributed to inducing the mobilization of the fatty acids from the adipose tissue triglyceride. On the other hand, it can be concluded that the insecticide did not affect protein metabolism at any dose level, so that the rate of protein degradation was balanced with the rate of synthesis. Moreover, absence of depressed serum protein levels means that oxamyl did not affect liver parenchymal cells responsible for synthesis of most serum proteins. Generally, in terms of what might be anticipated from such a compound with a low LD50, the data suggest that the maximum response of the studied parameters had not been reached, although higher doses are within the lethal range of oxamyl. Apparently it derives its toxic effect from being a potent anticholinesterase agent, but in general its acute toxicity to rats is of moderate order. In summary, the data reported herein is suggestive of a reversible toxic effect of oxamyl on the investigated parameters. These results suggest that further work, including histopathological examination as well as clinical tests, is necessary to elucidate the possibility of irreversible damage following oxamyl exposure. ACKNOWLEDGMENTS The authors acknowledge and thank Jerry Ito for statistical analysis and manuscript preparation.
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