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The effects of acute carbaryl exposure on clotting factor activity in the rat
ECOTOXICOLOCY
AND
ENVIRONMENTAL
SAFETY
(1984)
8,280-283
The Effects of Acute Carbaryl Exposure Clotting Factor Activity in the Rat
on
CHARLES D. Lox Department of Obstetrics and Gynecology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430 Received May 24, 1983 Male Sprague-Dawley rats were placed on a drinking solution containing either 10 parts per million (ppm) carbaryl or water for 30 days. Plasma was analyzed for the prothrombin time, partial thromboplastin time, fibrinogen, and clotting factor activity for coagulation factors II, V, VII, VIII, IX, X, XII and the platelet count. Only two hematological parameters measured were statistically different from the controls; these parameters were the platelet count and factor VII activity, both of which were reduced. Histological examination of the hepatic tissue illustrated that a number of pathological changes were occurring.
INTRODUCTION Due to the tremendous worldwide usageof insecticides for agricultural purposes, concern hasarisen asto potential health problems which could arise from the accidental exposure to thesechemicals. Although reports of acute toxicity following contamination from insecticides have appeared (Guthrie, 1980), much less is known about their subacute toxicity. Pathological changesassociatedwith insecticide exposure have also been reported (Deichman et al., 1969; Hodge et al., 1967; Kimbrough et al., 1971; Kuiper-Goodman et al., 1977; Sastry and Malik, 1982). However, little has been reported in terms of clotting activity studies following insecticide exposure. This is surprising in view of the fact that a number of the clotting factors (fibrinogen and factors II, V, VII, IX, and X) are synthesized in the liver (Ratnoff, 1977). Malathion or diazinon poisoning caused changes in clotting activity, including as short a time span as 2 hr following ingestion of these organophosphate compounds (Lox, 1982a,b, 1983). To ascertain if exposure to a prevalently used carbamate insecticide, carbaryl, would have any influence on hematologic function in the rat, the following experiment was taken. METHODS
AND
MATERIALS
Experimental design. Thirty male Sprague-Dawley rats between 250 and 300 g were utilized in this experiment. The rats were obtained from SASCO Animal Farms, Omaha, Nebraska. The rats were maintained in wire-mesh cages,2 per cage, under constant temperature (23°C) with 12 hr of light and 12 hr of dark daily throughout the experiment. Purina rat chow and drinking solutions were available ad libitum throughout the experiment. At the initiation of the experiment all animals were weighed and marked for identification. The rats were subsequently divided into two groups; 15 rats were placed on an ad libitum solution of tap water to act as controls. 0 147-65 13184 $3.00 Copyright Q 1984 by Academic Press. Inc. All rights of repmduction in any four rewed.
280
CARBARYL
281
AND COAGULATION
The second group of 15 rats was placed on an ad libitum drinking solution containing 10 ppm of the carbamate insecticide carbaryl (1-naphthyl-N-methylcarbamate), donated by Union Carbide Company, 99.8% purity. Each drinking solution bottle was filled with a known quantity of the appropriate drinking solution and daily intake of liquid was monitored throughout the experiment. The rats were allowed to drink either the water or the carbaryl for 30 days at which time the rats were sacrificed. All animals were sacrificed under ether anesthesia, with a 4.5-ml sample of sodium titrated (3.8%) whole blood being collected with a syringe from the dorsal aorta into siliconized tubes. Before blood sampling was initiated the animals were again weighed to determine body weight changes. At the time of sacrifice, liver tissue was also removed and placed in a solution of 10% Formalin for histological preparation. Plasma was collected by centrifugation (2500 rpm) at 4’C; this after an aliquot had been removed for the platelet count determination. The plasma was stored frozen at -20°C until analyzed, within 7 days. Analysis. The platelet count was determined manually using a hemocytometer (American Optical). Clotting activity was performed utilizing human factor-deficient substrate supplied by George Ring Biochemical, Wichita, Kansas. These included the prothrombin time (PT), partial thromboplastin time (APTT), fibrinogen, and coagulation factors II, V, VII, VIII, IX, X, and XII. As human deficient substrate was used, the percentage activity was determined against a normal human control for comparative purposes. Histological preparation. Following routine dehydration, the hepatic tissue was embedded in paralhn wax and subsequently cut at 6-pm thickness for staining with hematoxylin-eosin. Statistics. All data were analyzed for statistical significance utilizing Student’s t test. All data are considered to be statistically significant at the P < 0.05 level. RESULTS When compared to controls, those rats who were on the carbaryl drinking solution showed a decrease in voluntary intake of the carbaryl; however, this had no effect on body weight gain as can be seen in Table 1. In terms of clotting activity (Table 2), few changes were noted to occur following ingestion of the 10 ppm carbaryl solution. The two significantly different changes which were observed to occur were in the platelet count, which decreased, as did the factor VII clotting activity. TABLE
1
VOLUMESOFLIQUIDSINGESTEDANDBODY WEIGHT CHANGESIN MALE SPRAGUE-DAWLEYRATS DRINKING~ATEROR CARBARYLFOR~~ DAYS Control Total volume ingested (ml/2 rats) Body weight gain (g) Note. x + SD. * P < 0.05.
1103 f 141 81.9 + 21.4
Carbaryl 979* f 158 82.8 f 20.5
282
CHARLES
D. LOX
TABLE
EFFECTS OF
INGESTING
10 ppm IN MALE
2
CARBARYL FOR 30 DAYS ON CLOTTING SPRAGUE-DAWLEY RATS Percentage
(Z,
92
(mZ1)
II
(N =15)
13.8 + 0.6
Treatment
(N = 15)
13.9
& 0.5
35.2 + 3.6 34.3 + 4.6
153 106 + 19 + 16 143 96 + 13 + 19
activity
PLT (X 103/
v VII
Control
Acr~v~n
117 85 + 13 f 9 79 101* * 20 + 12
VIII
88 + 21 87 + 25
IX
x
102 +12 95 + 20
88 210 82 + 16
XII
mm”)
112 +11 102 f 19
672 + 150 513: k loo
Note. if f SD. * P < 0.05.
When compared to controls, microscopic evaluation of the liver tissue revealed a number of changeshad occurred in the liver of the rats who were drinking the carbaryl. This included some hepatocyte degeneration, central vein congestion, someleukocytic inhltration, and vacuolization of the cytoplasm. DISCUSSION The data obtained from this study tend to suggestthat drinking 10 ppm of a carbaryl solution by male rats can cause pathological changes to liver tissue as well as to the hemostatic mechanism. As thrombocytopenia was seento occur following the carbaryl treatment, as well as a decreasein factor VII activity, which is the first clotting factor activated in the extrinsic pathway, impairment of formation of the platelet plug following trauma conceivably might result. However, asneither the PT nor the APTT, both reflections of overall hemostasis, was abnormal, this suggeststhat as a whole the clotting cascade was not inhibited by the treatment. Many of the histological changes observed in this study were also observed by Sastry and Malik (1982) in fish that were exposed to diazinon. This supports the suggestion that insecticides can influence hepatic morphology, even at the low concentration used in this study. This was also reflected to some extent by the abnormal levels of factor VII activity, a protein which is synthesized in the liver. It has been shown that acute exposure to a diazinon concentration of 157 ppm for 14 days or 1750 ppm for 2 hr will cause significant changes in clotting activity (Lox, 1982b, 1983). This suggeststhat perhaps the ingestion of large concentrations of insecticide is required to establish significant disruptions in hematological function, if these chemicals are to act as a hepatic toxin in terms of hemostasis. As the concentration of carbaryl used in this study was considerably lessthan was the diazinon solution used in other studies, yet an effect on hepatic synthesized factor VII clotting activity was seen, suggeststhat low levels of carbaryl may have some effect on hemostasis. This could be of significance as the LDso of carbaryl has been shown to be somewhere between that of malathion and diazinon (Gaines, 1969). From the results of this study it appears that the carbamate insecticide carbaryl does have a possible deleterious effect on hemostasis,and certainly on hepatic morphology. It now needs to be seen whether carbaryl will induce changes following chronic low-dose exposure for long time spans.
CARBARYL
AND COAGULATION
283
ACKNOWLEDGMENT The author would like to acknowledge the kind donation of the carbaryl by Union Carbide Agricultural Products Division, Research Triangle Park, North Carolina.
REFERENCES DEICHMANN, W. B., KEPLINGER, N., DRESSLER,I., AND SALA, F. (1969). Retention of Dieldrin and DDT in the tissues of dogs fed aldrin and DDT individually and as a mixture. Toxicol. Appl. Pharmacol. 14, 205.
GAINES, T. B. (1969). Acute toxicity of pesticides. Toxicol. Appl. Pharrnacol. 14, 5 15. GUTHRIE, F. E. (1980). Pesticides in humans. In Introduction to Environmental Toxicology (Guthrie and Terry, eds.), pp. 289-312. Elsevier, New York. HODGE, H. C., BOYCE, A. M., DEICHMANN, W. B., AND KRAYBILL, H. F. (1967). Toxicology and noeffect levels of aldrin and dieldrin. Toxicol. Appl. Pharmacol. 10, 613. KIMBROUGH, R. D., GAINES, T. B., AND LINDER, R. E. (1971). The ultrastructure of livers of rats fed DDT and dieldrin. Arch. Environ. Health 22, 460. KUIPER-GOODMAN, T., GRANT, D. L., MOODIE, C. Z., KORSRUD, G. D., AND MUNROE, I. C. (1977). Subacute toxicity of hexachlorobenzene in the rat. Toxicol. Appl Pharmacol. 40, 529. Lox, C. D. (1982a). Short term malathion ingestion and blood clotting in the rat. J. Environ. Puthol. Toxicol. Oncol., in press. Lox, C. D. (1982b). The effects of short term diazinon exposure on blood clotting activity in the rat. J. Environ. Pathol. Toxicol. Oncol., in press. Lox, C. D. (1983). Effects of acute pesticide poisoning on blood clotting in the rat. Ecotoxicol. Environ. Saf 7, 45 l-454.
RATNOFF, 0. D. (1977). Blood clotting mechanisms: An overview. In Hemostasis: Biochemistry, Physiology and Pathology (Ogston and Bennett, eds.), pp. l-24. Wiley, New York. SASTRY,K. V., AND MALIK, P. V. (1982). Histopathological and enzymological alterations in the digestive system of a freshwater Teleost fish, Heteropneustes fossilis, exposed acutely and chronically to diazinon. Ecotoxicol.
Environ.
Saf: 6, 223.
AND
ENVIRONMENTAL
SAFETY
(1984)
8,280-283
The Effects of Acute Carbaryl Exposure Clotting Factor Activity in the Rat
on
CHARLES D. Lox Department of Obstetrics and Gynecology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430 Received May 24, 1983 Male Sprague-Dawley rats were placed on a drinking solution containing either 10 parts per million (ppm) carbaryl or water for 30 days. Plasma was analyzed for the prothrombin time, partial thromboplastin time, fibrinogen, and clotting factor activity for coagulation factors II, V, VII, VIII, IX, X, XII and the platelet count. Only two hematological parameters measured were statistically different from the controls; these parameters were the platelet count and factor VII activity, both of which were reduced. Histological examination of the hepatic tissue illustrated that a number of pathological changes were occurring.
INTRODUCTION Due to the tremendous worldwide usageof insecticides for agricultural purposes, concern hasarisen asto potential health problems which could arise from the accidental exposure to thesechemicals. Although reports of acute toxicity following contamination from insecticides have appeared (Guthrie, 1980), much less is known about their subacute toxicity. Pathological changesassociatedwith insecticide exposure have also been reported (Deichman et al., 1969; Hodge et al., 1967; Kimbrough et al., 1971; Kuiper-Goodman et al., 1977; Sastry and Malik, 1982). However, little has been reported in terms of clotting activity studies following insecticide exposure. This is surprising in view of the fact that a number of the clotting factors (fibrinogen and factors II, V, VII, IX, and X) are synthesized in the liver (Ratnoff, 1977). Malathion or diazinon poisoning caused changes in clotting activity, including as short a time span as 2 hr following ingestion of these organophosphate compounds (Lox, 1982a,b, 1983). To ascertain if exposure to a prevalently used carbamate insecticide, carbaryl, would have any influence on hematologic function in the rat, the following experiment was taken. METHODS
AND
MATERIALS
Experimental design. Thirty male Sprague-Dawley rats between 250 and 300 g were utilized in this experiment. The rats were obtained from SASCO Animal Farms, Omaha, Nebraska. The rats were maintained in wire-mesh cages,2 per cage, under constant temperature (23°C) with 12 hr of light and 12 hr of dark daily throughout the experiment. Purina rat chow and drinking solutions were available ad libitum throughout the experiment. At the initiation of the experiment all animals were weighed and marked for identification. The rats were subsequently divided into two groups; 15 rats were placed on an ad libitum solution of tap water to act as controls. 0 147-65 13184 $3.00 Copyright Q 1984 by Academic Press. Inc. All rights of repmduction in any four rewed.
280
CARBARYL
281
AND COAGULATION
The second group of 15 rats was placed on an ad libitum drinking solution containing 10 ppm of the carbamate insecticide carbaryl (1-naphthyl-N-methylcarbamate), donated by Union Carbide Company, 99.8% purity. Each drinking solution bottle was filled with a known quantity of the appropriate drinking solution and daily intake of liquid was monitored throughout the experiment. The rats were allowed to drink either the water or the carbaryl for 30 days at which time the rats were sacrificed. All animals were sacrificed under ether anesthesia, with a 4.5-ml sample of sodium titrated (3.8%) whole blood being collected with a syringe from the dorsal aorta into siliconized tubes. Before blood sampling was initiated the animals were again weighed to determine body weight changes. At the time of sacrifice, liver tissue was also removed and placed in a solution of 10% Formalin for histological preparation. Plasma was collected by centrifugation (2500 rpm) at 4’C; this after an aliquot had been removed for the platelet count determination. The plasma was stored frozen at -20°C until analyzed, within 7 days. Analysis. The platelet count was determined manually using a hemocytometer (American Optical). Clotting activity was performed utilizing human factor-deficient substrate supplied by George Ring Biochemical, Wichita, Kansas. These included the prothrombin time (PT), partial thromboplastin time (APTT), fibrinogen, and coagulation factors II, V, VII, VIII, IX, X, and XII. As human deficient substrate was used, the percentage activity was determined against a normal human control for comparative purposes. Histological preparation. Following routine dehydration, the hepatic tissue was embedded in paralhn wax and subsequently cut at 6-pm thickness for staining with hematoxylin-eosin. Statistics. All data were analyzed for statistical significance utilizing Student’s t test. All data are considered to be statistically significant at the P < 0.05 level. RESULTS When compared to controls, those rats who were on the carbaryl drinking solution showed a decrease in voluntary intake of the carbaryl; however, this had no effect on body weight gain as can be seen in Table 1. In terms of clotting activity (Table 2), few changes were noted to occur following ingestion of the 10 ppm carbaryl solution. The two significantly different changes which were observed to occur were in the platelet count, which decreased, as did the factor VII clotting activity. TABLE
1
VOLUMESOFLIQUIDSINGESTEDANDBODY WEIGHT CHANGESIN MALE SPRAGUE-DAWLEYRATS DRINKING~ATEROR CARBARYLFOR~~ DAYS Control Total volume ingested (ml/2 rats) Body weight gain (g) Note. x + SD. * P < 0.05.
1103 f 141 81.9 + 21.4
Carbaryl 979* f 158 82.8 f 20.5
282
CHARLES
D. LOX
TABLE
EFFECTS OF
INGESTING
10 ppm IN MALE
2
CARBARYL FOR 30 DAYS ON CLOTTING SPRAGUE-DAWLEY RATS Percentage
(Z,
92
(mZ1)
II
(N =15)
13.8 + 0.6
Treatment
(N = 15)
13.9
& 0.5
35.2 + 3.6 34.3 + 4.6
153 106 + 19 + 16 143 96 + 13 + 19
activity
PLT (X 103/
v VII
Control
Acr~v~n
117 85 + 13 f 9 79 101* * 20 + 12
VIII
88 + 21 87 + 25
IX
x
102 +12 95 + 20
88 210 82 + 16
XII
mm”)
112 +11 102 f 19
672 + 150 513: k loo
Note. if f SD. * P < 0.05.
When compared to controls, microscopic evaluation of the liver tissue revealed a number of changeshad occurred in the liver of the rats who were drinking the carbaryl. This included some hepatocyte degeneration, central vein congestion, someleukocytic inhltration, and vacuolization of the cytoplasm. DISCUSSION The data obtained from this study tend to suggestthat drinking 10 ppm of a carbaryl solution by male rats can cause pathological changes to liver tissue as well as to the hemostatic mechanism. As thrombocytopenia was seento occur following the carbaryl treatment, as well as a decreasein factor VII activity, which is the first clotting factor activated in the extrinsic pathway, impairment of formation of the platelet plug following trauma conceivably might result. However, asneither the PT nor the APTT, both reflections of overall hemostasis, was abnormal, this suggeststhat as a whole the clotting cascade was not inhibited by the treatment. Many of the histological changes observed in this study were also observed by Sastry and Malik (1982) in fish that were exposed to diazinon. This supports the suggestion that insecticides can influence hepatic morphology, even at the low concentration used in this study. This was also reflected to some extent by the abnormal levels of factor VII activity, a protein which is synthesized in the liver. It has been shown that acute exposure to a diazinon concentration of 157 ppm for 14 days or 1750 ppm for 2 hr will cause significant changes in clotting activity (Lox, 1982b, 1983). This suggeststhat perhaps the ingestion of large concentrations of insecticide is required to establish significant disruptions in hematological function, if these chemicals are to act as a hepatic toxin in terms of hemostasis. As the concentration of carbaryl used in this study was considerably lessthan was the diazinon solution used in other studies, yet an effect on hepatic synthesized factor VII clotting activity was seen, suggeststhat low levels of carbaryl may have some effect on hemostasis. This could be of significance as the LDso of carbaryl has been shown to be somewhere between that of malathion and diazinon (Gaines, 1969). From the results of this study it appears that the carbamate insecticide carbaryl does have a possible deleterious effect on hemostasis,and certainly on hepatic morphology. It now needs to be seen whether carbaryl will induce changes following chronic low-dose exposure for long time spans.
CARBARYL
AND COAGULATION
283
ACKNOWLEDGMENT The author would like to acknowledge the kind donation of the carbaryl by Union Carbide Agricultural Products Division, Research Triangle Park, North Carolina.
REFERENCES DEICHMANN, W. B., KEPLINGER, N., DRESSLER,I., AND SALA, F. (1969). Retention of Dieldrin and DDT in the tissues of dogs fed aldrin and DDT individually and as a mixture. Toxicol. Appl. Pharmacol. 14, 205.
GAINES, T. B. (1969). Acute toxicity of pesticides. Toxicol. Appl. Pharrnacol. 14, 5 15. GUTHRIE, F. E. (1980). Pesticides in humans. In Introduction to Environmental Toxicology (Guthrie and Terry, eds.), pp. 289-312. Elsevier, New York. HODGE, H. C., BOYCE, A. M., DEICHMANN, W. B., AND KRAYBILL, H. F. (1967). Toxicology and noeffect levels of aldrin and dieldrin. Toxicol. Appl. Pharmacol. 10, 613. KIMBROUGH, R. D., GAINES, T. B., AND LINDER, R. E. (1971). The ultrastructure of livers of rats fed DDT and dieldrin. Arch. Environ. Health 22, 460. KUIPER-GOODMAN, T., GRANT, D. L., MOODIE, C. Z., KORSRUD, G. D., AND MUNROE, I. C. (1977). Subacute toxicity of hexachlorobenzene in the rat. Toxicol. Appl Pharmacol. 40, 529. Lox, C. D. (1982a). Short term malathion ingestion and blood clotting in the rat. J. Environ. Puthol. Toxicol. Oncol., in press. Lox, C. D. (1982b). The effects of short term diazinon exposure on blood clotting activity in the rat. J. Environ. Pathol. Toxicol. Oncol., in press. Lox, C. D. (1983). Effects of acute pesticide poisoning on blood clotting in the rat. Ecotoxicol. Environ. Saf 7, 45 l-454.
RATNOFF, 0. D. (1977). Blood clotting mechanisms: An overview. In Hemostasis: Biochemistry, Physiology and Pathology (Ogston and Bennett, eds.), pp. l-24. Wiley, New York. SASTRY,K. V., AND MALIK, P. V. (1982). Histopathological and enzymological alterations in the digestive system of a freshwater Teleost fish, Heteropneustes fossilis, exposed acutely and chronically to diazinon. Ecotoxicol.
Environ.
Saf: 6, 223.