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Effects of acute pesticide poisoning on blood clotting in the rat
ECOTOXICOLOGY
AND
ENVIRONMENTAL
SAFETY
7,45
1-454
Effects of Acute Pesticide Poisoning CHARLES Department
of Obstetrics
and Gynecology,
Texas
Received
(1983)
on Blood Clotting in the Rat
D. Lox Tech Health
October
Sciences
Center.
Lubbock,
Texas
79430
14, 1982
Male Sprague-Dawley rats were treated with I ml of diazinon or malathion, 1750 ppm given orally by gavage. Clotting determinations included the prothrombin time, partial thromboplastin time, fibrinogen, and coagulation factors II, V, VII, and X, plus the hematocrit and platelet count. The results suggest that insecticides influence the clotting times of rats ingesting these insecticides even after as short a time span as 2 hr. INTRODUCTION
The use of pesticides is extensive throughout the world, and problems resulting from insecticide toxicity have been reported to occur in the industrial worker, formulator, applicator and nonapplicator agricultural worker, and other accidental exposures (Guthrie, 1980). Many of the reports dealt with individuals who were exposed to low levels of insecticides over periods of time or who had sudden exposure to a massive level of the insecticide, usually by inhalation or dermal contact. Morbidity and mortality statistics for insecticide poisoning are much less prevalent, however. It has been reported that the incidence of pesticide mortalities has been decreasing in the United States, with 100 reported in 1961 and 33 reported in 1974. Included in these deaths were exposures to the insecticides diazinon, malathion, and carbaryl (Hayes and Vaughn, 1978). In Rhodesia, reported organophosphate poisonings covering the period 1974 to 1978 totaled 105. Of these, 44 were attempted suicides. 16 of which had fatal consequences (Hayes et al., 1980). In Nebraska, 14 individuals who ate contaminated cucumbers demonstrated pesticide intoxication from the carbamate insecticide aldicarb (Goes et al., 1980). Statistics from Arizona for pesticideassociated hospitalizations for the years 197 1 to 1973 indicated 159 occupationally related admissions, 42 nonoccupational admissions, 6 attempted suicides, and 35 admissions in children less than 4 years of age (Alcorn et al., 1980). Much less is known about the effects of pesticide poisoning on hepatic function. It has been reported to cause liver necrosis and hypertrophy, histological changes, fatty infiltration, and atypical mitochondria (Kuiper-Goodman et al., 1977; Kimbrough et al., 197 1; Hodge et al., 1976; Deichman et al,, 1969; Sastry and Malik, 1982.) Reports of possible changes in the clotting mechanisms due to insecticide poisoning are scarce, with only one known report in the literature (Lox, 1982). As many of the blood-clotting proteins are synthesized in the liver (Ratnoff, 1977), where the pesticides are detoxified, this seems an important area to investigate. This could be especially important in acute massive doses following oral pesticide poisoning. METHODS
AND
MATERIALS
Male Sprague-Dawley rats, approximately 400 g each, were used in this experiment. All animals were housed in a rat colony, two to a cage, with alternating 12-hr dark 451
0 147-65 Copyright All n&s
13183
$3.00
0 1983 by Academtc Press., Inc. uf reproduction in any form reserved.
452
CHARLES
D. LOX
and light cycles. Drinking water and Purina Rat Chow were available ad libidum throughout the experiment. Eighteen rats were anesthetized and a l-ml sample of sodium titrated (3.8%) whole blood was taken via cardiac puncture. Each animal thus acted as its own control. One hour following the cardiac puncture, the animals (nine in each group) were given a l.O-ml solution via gavage of water containing a suspension of malathion or diazinon, at a concentration of 1750 ppm. Malathion [(dimethoxyphosphinothioyl)thiobutanedioic acid diethyl ester], 99% pure, Chem Service, Inc. (West Chester, Pa.), and diazinon (0,Odimethyl O-2-isopropyl-4-methyl6-pyrimidinyl thiophosphate), 87.6% pure, Ciba-Geigy Corporation (Greensboro, N.C.), were used. Two hours following the gavage treatment the animals were sacrificed. All animals were sacrificed under ether anesthesia and a 4.5-ml sample of sodium titrated (3.8%) whole blood was collected from the aorta. Plasma samples were analyzed for hematocrit using a Clay-Adams Autocrit II. Platelet counts were determined manually using a hemocytometer (American Optical). Clotting times for the following coagulation factors were measured on a COAG-AMATE 2001 coagulation timer (General Diagnostics Corp.), using human factordeficient substrates supplied by George Ring Biochemical, Wichita, Kansas. These included the prothrombin time (PT), the partial thromboplastin time (APTT), and the following hepatic-synthesized clotting factor activities: fibrinogen (FIB) and factors II, V, VII, and X. All values for these factors are reported in seconds required for a clot to form spectrophotometrically when the test plasma is added to factor-deficient substrate. Statistical analysis of the data was performed utilizing the paired t test. All significant differences are at the 95% confidence interval, P < 0.05. RESULTS Changes in blood-clotting times following 1 ml of diazinon or malathion given by gavage can be seen in Table 1. As is evidenced, when the animal was subjected to the insecticides even for as short a time span as 2 hr there were significant changes TABLE CHANGES
IN CLOTTING TIMES BY GAVAGE
1
2 hr FOLLOWING IN MALE RATS
1750 ppm OF INSECTICIDE (X f SD)
Seconds
Malathion Pregavage Postgavage Diazinon Pregavage Postgavage
* P < 0.05.
PT
APTT
FIB
II
V
VII
x
HCT (a)
13.8 kO.1 13.1 * 0.2*
31.8 f 2.9 27.7 f 3.0
9.6 + 0.5 10.1 + 0.3
16.6 + 0.5 16.5 f 0.5
15.3 * 0.3 15.5 f 0.2
17.4 f 0.4 17.9 + 0.4
29.1 + 0.7 33.7 + 0.9;
41.1 + 3.0 31.9 + 3.4*
14.4 AZ 0.4 13.4 + 0.1*
31.3 + 1.0 27.0 + 0.9*
9.6 + 0.4 10.8 f 0.7*
16.8 + 0.5 15.8 + 0.3*
16.3 * 0.1 14.7 * 0.3.
17.1 + 0.2 16.9 f 0.4
30.5 + 1.2 27.1 2 3.0*
40.9 + 1.1 38.2 + 3.0
PLT (X 103/mm3)
940 133 440 + 120*
f
856 f 61 694 f 140*
INSECTICIDES
453
AND COAGULATION
in some of the blood-clotting parameters measured, suggesting a rapid and demonstrable effect of insecticides on the coagulation system, including those which are hepatic in origin. DISCUSSION As can be seen in Table 1, a 2-hr exposure to insecticides by gavage had an effect on the clotting factor activity. As diazinon on a milligram to milligram basis is a more potent insecticide in terms of the LDSo in the rat than is malathion [Gaines, 19691, it is not surprising that diazinon had a greater effect on the clotting parameters measured than did malathion. In both treatments the platelet counts were abnormal following a 2-hr exposure to the insecticide, both being reduced, as was the hematocrit following malathion. This malathion-induced decrease in hematocrit seems extremely rapid for a reduction of the red blood cell volume to occur. Of the hepatic-synthesized clotting factors, diazinon affected four of the five measured. Three of these four were accelerated (II, V, and X) while the fibrinogen time was prolonged. These accelerated factors, part of the common pathway, when coupled with the accelerated PT and APTT in the diazinon-treated animal, would tend to suggest that an overall condition of hypercoagulability existed. Likewise, the malathion-treated animals also had accelerated FT. The data of this experiment are interesting in light of the report of Poklis (1980) who reported that a 54-year-old female who committed suicide by ingesting diazinon had demonstrable petechial hemorrages throughout the stomach and gastric mucosa as well as in the brain tissue. Tissue diazinon levels were detected throughout the body as well as in the bloodstream and bile. The presence of these petechia are suggestive of a hemorraghic disorder, which conceivably could be related to an alteration in coagulation due to the ingestion of this insecticide. The data from this study suggest that insecticides might affect hemostasis in general. This could have significance in individuals who have ingested insecticides orally as in cases of attempted suicide or accidental intake by infants. Preliminary data from this experiment also suggest that insecticides circulate rapidly through the body and can have a rapid and definitive effect on the synthesis or the activity of the bloodclotting factors. ACKNOWLEDGMENT The author wishes to thank Ciba-Geigy Corporation for the donations of diazinon.
REFERENCES ALCORN, E. P., ANGEL, E., AND HUGHES, J. H. (1980). Pesticides in Arizona, an update. Ariz. Med. 37, 36.
DEICHMANN, W. B., KEPLINGER, N., DRESSLER,I., AND SALA, F. (1969). Retention of Dieldrin and DDT in the tissues of dogs fed AIdrin and DDT individually and as a mixture. Toxicol. Appl. Phormacol. 14, 205.
GOES, T. B. (1969). Acute toxicity of pesticides. Toxicol. Appl. Pharmacol. 14, 513. GOES, E. A., SAVAGE, E. P., GIBBONS, G., AARONSON, M., AND FORD, S. A. (1980). Suspected foodhom carhamate pesticide intoxication associated with ingestion of hydroponic cucumbers. Amer. J. Epidemiol. 111, 254.
GUTHRIE, F. E., (1980). Pesticides in humans. In Introduction Terry, eds.), pp. 289-312, Elsevier, New York.
to Environmental
Toxicology
(Guthrie and
454
CHARLES
D. LOX
HAYES, W. J., AND VAUGHN, W. K. (1977). Mortality from Pesticides in the United States in 1973 and 1974. Toxicol. Appl. Pharmacol. 42, 235. HAYES, N. M., VAN DER WESTHUIZEN, N. G., AND GELFAND, M. (1978). Organophosphate poisoning in Rhodesia. A study of the clinical features and management of 105 patients. S. Afr. Med. J. 54,230. 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 Die&in. Arch. Envir. Health 22, 460. KUIPER-GOODMAN, T., GRANT, D. L., MOODIE, C. A., KORSRUD, G. D., AND MUNROE, I. C. (1977). Subacute toxicity of hexachlorobenzene in the rat. Toxicol. Appl. Pharrnacol. 40, 529. Lox, C. D. (1982). Short-term malathion ingestion and blood clotting in the rat .I. Envir. Path. Toxicol., in press. POKLIS, A., KUTZ, F. W., SPERLING, J. F., AND MORGAN, D. T. (1980). A fatal diazinon poisoning. Forensic Sci. Int. 15, 135. RATNOFF, 0. D. ( 1977). Blood clotting mechanisms: An overview. In Haemostasis Siocherkstry, Physiology and Pathology (Ogston and Bennett, eds.), pp. l-24, Wiley, London. 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. Safety 6, 223.
AND
ENVIRONMENTAL
SAFETY
7,45
1-454
Effects of Acute Pesticide Poisoning CHARLES Department
of Obstetrics
and Gynecology,
Texas
Received
(1983)
on Blood Clotting in the Rat
D. Lox Tech Health
October
Sciences
Center.
Lubbock,
Texas
79430
14, 1982
Male Sprague-Dawley rats were treated with I ml of diazinon or malathion, 1750 ppm given orally by gavage. Clotting determinations included the prothrombin time, partial thromboplastin time, fibrinogen, and coagulation factors II, V, VII, and X, plus the hematocrit and platelet count. The results suggest that insecticides influence the clotting times of rats ingesting these insecticides even after as short a time span as 2 hr. INTRODUCTION
The use of pesticides is extensive throughout the world, and problems resulting from insecticide toxicity have been reported to occur in the industrial worker, formulator, applicator and nonapplicator agricultural worker, and other accidental exposures (Guthrie, 1980). Many of the reports dealt with individuals who were exposed to low levels of insecticides over periods of time or who had sudden exposure to a massive level of the insecticide, usually by inhalation or dermal contact. Morbidity and mortality statistics for insecticide poisoning are much less prevalent, however. It has been reported that the incidence of pesticide mortalities has been decreasing in the United States, with 100 reported in 1961 and 33 reported in 1974. Included in these deaths were exposures to the insecticides diazinon, malathion, and carbaryl (Hayes and Vaughn, 1978). In Rhodesia, reported organophosphate poisonings covering the period 1974 to 1978 totaled 105. Of these, 44 were attempted suicides. 16 of which had fatal consequences (Hayes et al., 1980). In Nebraska, 14 individuals who ate contaminated cucumbers demonstrated pesticide intoxication from the carbamate insecticide aldicarb (Goes et al., 1980). Statistics from Arizona for pesticideassociated hospitalizations for the years 197 1 to 1973 indicated 159 occupationally related admissions, 42 nonoccupational admissions, 6 attempted suicides, and 35 admissions in children less than 4 years of age (Alcorn et al., 1980). Much less is known about the effects of pesticide poisoning on hepatic function. It has been reported to cause liver necrosis and hypertrophy, histological changes, fatty infiltration, and atypical mitochondria (Kuiper-Goodman et al., 1977; Kimbrough et al., 197 1; Hodge et al., 1976; Deichman et al,, 1969; Sastry and Malik, 1982.) Reports of possible changes in the clotting mechanisms due to insecticide poisoning are scarce, with only one known report in the literature (Lox, 1982). As many of the blood-clotting proteins are synthesized in the liver (Ratnoff, 1977), where the pesticides are detoxified, this seems an important area to investigate. This could be especially important in acute massive doses following oral pesticide poisoning. METHODS
AND
MATERIALS
Male Sprague-Dawley rats, approximately 400 g each, were used in this experiment. All animals were housed in a rat colony, two to a cage, with alternating 12-hr dark 451
0 147-65 Copyright All n&s
13183
$3.00
0 1983 by Academtc Press., Inc. uf reproduction in any form reserved.
452
CHARLES
D. LOX
and light cycles. Drinking water and Purina Rat Chow were available ad libidum throughout the experiment. Eighteen rats were anesthetized and a l-ml sample of sodium titrated (3.8%) whole blood was taken via cardiac puncture. Each animal thus acted as its own control. One hour following the cardiac puncture, the animals (nine in each group) were given a l.O-ml solution via gavage of water containing a suspension of malathion or diazinon, at a concentration of 1750 ppm. Malathion [(dimethoxyphosphinothioyl)thiobutanedioic acid diethyl ester], 99% pure, Chem Service, Inc. (West Chester, Pa.), and diazinon (0,Odimethyl O-2-isopropyl-4-methyl6-pyrimidinyl thiophosphate), 87.6% pure, Ciba-Geigy Corporation (Greensboro, N.C.), were used. Two hours following the gavage treatment the animals were sacrificed. All animals were sacrificed under ether anesthesia and a 4.5-ml sample of sodium titrated (3.8%) whole blood was collected from the aorta. Plasma samples were analyzed for hematocrit using a Clay-Adams Autocrit II. Platelet counts were determined manually using a hemocytometer (American Optical). Clotting times for the following coagulation factors were measured on a COAG-AMATE 2001 coagulation timer (General Diagnostics Corp.), using human factordeficient substrates supplied by George Ring Biochemical, Wichita, Kansas. These included the prothrombin time (PT), the partial thromboplastin time (APTT), and the following hepatic-synthesized clotting factor activities: fibrinogen (FIB) and factors II, V, VII, and X. All values for these factors are reported in seconds required for a clot to form spectrophotometrically when the test plasma is added to factor-deficient substrate. Statistical analysis of the data was performed utilizing the paired t test. All significant differences are at the 95% confidence interval, P < 0.05. RESULTS Changes in blood-clotting times following 1 ml of diazinon or malathion given by gavage can be seen in Table 1. As is evidenced, when the animal was subjected to the insecticides even for as short a time span as 2 hr there were significant changes TABLE CHANGES
IN CLOTTING TIMES BY GAVAGE
1
2 hr FOLLOWING IN MALE RATS
1750 ppm OF INSECTICIDE (X f SD)
Seconds
Malathion Pregavage Postgavage Diazinon Pregavage Postgavage
* P < 0.05.
PT
APTT
FIB
II
V
VII
x
HCT (a)
13.8 kO.1 13.1 * 0.2*
31.8 f 2.9 27.7 f 3.0
9.6 + 0.5 10.1 + 0.3
16.6 + 0.5 16.5 f 0.5
15.3 * 0.3 15.5 f 0.2
17.4 f 0.4 17.9 + 0.4
29.1 + 0.7 33.7 + 0.9;
41.1 + 3.0 31.9 + 3.4*
14.4 AZ 0.4 13.4 + 0.1*
31.3 + 1.0 27.0 + 0.9*
9.6 + 0.4 10.8 f 0.7*
16.8 + 0.5 15.8 + 0.3*
16.3 * 0.1 14.7 * 0.3.
17.1 + 0.2 16.9 f 0.4
30.5 + 1.2 27.1 2 3.0*
40.9 + 1.1 38.2 + 3.0
PLT (X 103/mm3)
940 133 440 + 120*
f
856 f 61 694 f 140*
INSECTICIDES
453
AND COAGULATION
in some of the blood-clotting parameters measured, suggesting a rapid and demonstrable effect of insecticides on the coagulation system, including those which are hepatic in origin. DISCUSSION As can be seen in Table 1, a 2-hr exposure to insecticides by gavage had an effect on the clotting factor activity. As diazinon on a milligram to milligram basis is a more potent insecticide in terms of the LDSo in the rat than is malathion [Gaines, 19691, it is not surprising that diazinon had a greater effect on the clotting parameters measured than did malathion. In both treatments the platelet counts were abnormal following a 2-hr exposure to the insecticide, both being reduced, as was the hematocrit following malathion. This malathion-induced decrease in hematocrit seems extremely rapid for a reduction of the red blood cell volume to occur. Of the hepatic-synthesized clotting factors, diazinon affected four of the five measured. Three of these four were accelerated (II, V, and X) while the fibrinogen time was prolonged. These accelerated factors, part of the common pathway, when coupled with the accelerated PT and APTT in the diazinon-treated animal, would tend to suggest that an overall condition of hypercoagulability existed. Likewise, the malathion-treated animals also had accelerated FT. The data of this experiment are interesting in light of the report of Poklis (1980) who reported that a 54-year-old female who committed suicide by ingesting diazinon had demonstrable petechial hemorrages throughout the stomach and gastric mucosa as well as in the brain tissue. Tissue diazinon levels were detected throughout the body as well as in the bloodstream and bile. The presence of these petechia are suggestive of a hemorraghic disorder, which conceivably could be related to an alteration in coagulation due to the ingestion of this insecticide. The data from this study suggest that insecticides might affect hemostasis in general. This could have significance in individuals who have ingested insecticides orally as in cases of attempted suicide or accidental intake by infants. Preliminary data from this experiment also suggest that insecticides circulate rapidly through the body and can have a rapid and definitive effect on the synthesis or the activity of the bloodclotting factors. ACKNOWLEDGMENT The author wishes to thank Ciba-Geigy Corporation for the donations of diazinon.
REFERENCES ALCORN, E. P., ANGEL, E., AND HUGHES, J. H. (1980). Pesticides in Arizona, an update. Ariz. Med. 37, 36.
DEICHMANN, W. B., KEPLINGER, N., DRESSLER,I., AND SALA, F. (1969). Retention of Dieldrin and DDT in the tissues of dogs fed AIdrin and DDT individually and as a mixture. Toxicol. Appl. Phormacol. 14, 205.
GOES, T. B. (1969). Acute toxicity of pesticides. Toxicol. Appl. Pharmacol. 14, 513. GOES, E. A., SAVAGE, E. P., GIBBONS, G., AARONSON, M., AND FORD, S. A. (1980). Suspected foodhom carhamate pesticide intoxication associated with ingestion of hydroponic cucumbers. Amer. J. Epidemiol. 111, 254.
GUTHRIE, F. E., (1980). Pesticides in humans. In Introduction Terry, eds.), pp. 289-312, Elsevier, New York.
to Environmental
Toxicology
(Guthrie and
454
CHARLES
D. LOX
HAYES, W. J., AND VAUGHN, W. K. (1977). Mortality from Pesticides in the United States in 1973 and 1974. Toxicol. Appl. Pharmacol. 42, 235. HAYES, N. M., VAN DER WESTHUIZEN, N. G., AND GELFAND, M. (1978). Organophosphate poisoning in Rhodesia. A study of the clinical features and management of 105 patients. S. Afr. Med. J. 54,230. 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 Die&in. Arch. Envir. Health 22, 460. KUIPER-GOODMAN, T., GRANT, D. L., MOODIE, C. A., KORSRUD, G. D., AND MUNROE, I. C. (1977). Subacute toxicity of hexachlorobenzene in the rat. Toxicol. Appl. Pharrnacol. 40, 529. Lox, C. D. (1982). Short-term malathion ingestion and blood clotting in the rat .I. Envir. Path. Toxicol., in press. POKLIS, A., KUTZ, F. W., SPERLING, J. F., AND MORGAN, D. T. (1980). A fatal diazinon poisoning. Forensic Sci. Int. 15, 135. RATNOFF, 0. D. ( 1977). Blood clotting mechanisms: An overview. In Haemostasis Siocherkstry, Physiology and Pathology (Ogston and Bennett, eds.), pp. l-24, Wiley, London. 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. Safety 6, 223.