- Email: [email protected]
Target site bioactivation of the neurotoxic organophosphorus insecticide parathion in partially hepatectomized rats
Life Sciences, Vol. 48, pp. 1023-1029 Printed in the U.S.A.
Pergamon Press
TARGET SITE BIOACTIVATION OF THE NEUROTOXIC ORGANOPHOSPHORUS INSECTICIDE PARATHION IN PARTIALLY HEPATECTOMIZED RATS Janice E. Chambers I, Howard W. Chambers and John E. Snawder 2 Depts. of Biological Sciences and Entomology, Mississippi State University, Mississippi State, Mississippi (Received in final form January 7, 1991) Summary Target organ bioactivation of phosphorothionate insecticides to their potent anticholinesterase oxon metabolites (for example, parathion to paraoxon) may be extremely important in toxicity because liver and blood provide so much potential protection by a variety of mechanisms, such as the aliesterases which serve as alternate phosphorylation sites. To determine whether the brain can produce sufficient oxon in vivo to contribute to toxicity, male rats were partially hepatectomized and injected i.v. with 1.5 mg/kg parathion. After 30 minutes, brain AChE was inhibited 68% whereas liver and plasma aliesterases were unaffected. Because aliesterases are far more sensitive to paraoxon inhibition than is brain ACHE, these results indicate that neither the liver nor extra-hepatic tissues were contributing oxon into the blood stream. Thus target site activation of parathion occurred in vivo at sufficient levels to contribute substantially to toxicity. Many xenobiotics require bioactivation to display toxicity because toxic effects are mediated by covalent binding between a reactive metabolite and the target molecule. The liver is large and has very high activities of the xenobiotic metabolizing enzymes which are responsible for bioactivation reactions, usually cytochrome P-450-dependent monooxygenases (P-450). In the case of non-hepatotoxic xenobiotics, however, these metabolites will need to react with target molecules distant from the liver, thus requiring transport. It seems unlikely that appreciable quantities of hepatically-generated reactive metabolite would be able to reach the target tissue intact if very many alternative reactive sites exist in either the liver or the blood stream. In such cases, it may be a criterion for toxicity that bioactivation of the xenobiotic occur within the target tissue so that formation of very labile metabolites would occur in close proximity to the target molecules. The above scenario is quite likely for phosphorothionate insecticides, the most common chemical form of organophosphorus insecticides. The phosphorothionate is metabolized by P-450 to a potent anticholinesterase, the oxon or phosphate; for example, parathion is converted to paraoxon (i). The bioactivation increases the anticholinesterase potency by about three orders of magnitude (2). The phosphorylation of the serine hydroxyl at the active site of acetylcholinesterase (ACHE) within the nervous system is currently IAddress correspondence to: Department of Biological Sciences, Mississippi State University, P.O. Drawer GY, Mississippi State, MS 39762, USA. ZPresent address: National Center for Toxicological Research, Jefferson, AR. 0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc
1024
Target Organ Bioactlvation of Parathion
Vol. 48, No. I0, 1991
accepted as the mechanism of acute toxicity (4). However, the oxons react not only with AChE but also with other serlne esterases and with serine proteases. Phosphorylation of these non-target serine esterases/proteases serves to stoichiometrically destroy the oxon. Enzymes that can participate in this non-catalytlc detoxication process are hepatic aliesterases (carboxylesterases), plasma allesterases and butyrylcholinesterase, erythrocyte ACHE, and serine proteases in a variety of tissues. The high enzyme activities, implying high concentrations, of aliesterases in liver and plasma suggest that they may be particularly important in protecting the organism from intoxication by organophosphates. Selective inhibition of the allesterases in vivo resulted in enhanced toxicity of the organephosphate paraoxon (5). The allesterases have been implicated as alternate phosphorylation sites in poisoning by the organophosphate nerve agents (6-8). Because the hepatic aliesterases are about an order of magnitude more sensitive than brain ACHE, they are particularly likely to sequester oxon as it is generated within the liver (9). In fact, the liver effectively retains most of the parathion perfused into it with little paraoxon observed escaping from the perfused liver (i0), indicating that the liver is a very effective trap for any paraoxon it generates. Thus, only low quantities of oxon should be expected to escape the liver unless the dose were so high as to overwhelm hepatic protective mechanisms. What little escapes could then be sequestered by the protective factors in the plasma. Target organ activation, then, may be responsible for generating most of the oxon which inhibits nervous system ACHE. The brain possesses a variety of cytochrome P-450-dependent monooxygenase activities (11-15). Both the microsomal and mitochondrial fractions of brain possess desulfuration activities for six phosphorothionates (2,3,16). In these studies there was a very good correlation between in vitro brain desulfuration activity and acute toxicity level, whereas there was no correlation between liver desulfuration activity and toxicity. This relationship suggested that brain desulfuration activity may have generated the oxon responsible for toxicity and that the level of brain activation was an important determinant of acute toxicity level. Nevertheless, the brain desulfuration activity is extremely low compared with the liver, so the question remains as to whether the brain could actually generate significant amounts of oxon. Experiments were conducted in the rat following ligation of the descending aorta, thus removing the liver from the circulation of the brain. In this protocol with an extremely high dose of parathion and a 15 minute time frame, a significant amount of brain AChE inhibition occurred (17). The short time frame was necessitated by the large amount of the body (about 60%) which had been removed from the circulation, and the extremely high dose was necessitated by the short time frame. This paper describes a protocol involving partial hepatectomy which allowed a lower dose and longer time frame to be employed to mimic more closely the conditions of a realistic phosphorothionate intoxication. As with the previous ligation experiments (17), brain AChE inhibition was assessed following parathion administration. Also assessed was the inhibition of hepatic and plasma aliesterases, which are an order of magnitude more sensitive to paraoxon than brain ACHE, and therefore provide a highly sensitive indicator of the presence of paraoxon. Materials and Methods Animals: Male Sprague-Dawley derived rats [CrI:CD(SD)BR] from an original Charles River stock weighing about 400-450 g were used. Before use, they were housed in a temperature-controlled animal room with a 12:12 hr light cycle and with Purina laboratory rodent food and tap water freely available.
Vol. 48, No. I0, 1991
Target Organ Bioactivation
of Parathion
1025
Chemicals: Parathion was a gift of Monsanto Chemical Company (St. Louis, MO) and was purified by recrystalllzatlon from methanol. Chemicals for enzyme assays were obtained from Sigma Chemical Company (St. Louis, MO). Surgical procedure and treatment: Rats were anesthetized with methoxyflurane and were maintained throughout surgery under anesthesia. All surgical procedures were conducted in a fume hood. The animal's abdomen was shaved and an incision was made to expose the liver. All lobes of the liver except the right lateral lobe and the caudal lobe, through which the posterior vena cava courses, were removed following llgation of the lobe at its base. Removal of these two remaining lobes would have required much more prolonged and disruptive surgery than was employed. This procedure removed about 70% (6872%) of the liver mass. The liver removed was chilled for use in a preinjection allesterase determination. The incision in the abdominal muscle wall was closed with sutures and the skin was closed with wound clips. Sham operations were also performed. Prior to recovery from anesthesia, treatment with 1.5 mg/kg parathion in propylene glycol was administered as an i.v. injection into the tail vein in a volume of 0.5 ml/kg. Propylene glycol in the same volume was administered to controls. Prior to injection the end of the tail was cut to obtain a blood sample which was collected into a 0.4 ml microcentrifuge tube containing a few mg of solid EDTA as an anticoagulant. The blood was gently mixed with the EDTA and the sample was chilled. Plasma was obtained by centrifugation at room temperature in an Eppendorf microcentrifuge at 17,000 g for 5 min. Each animal was allowed to recover from anesthesia. At the appropriate time each was euthanitized by decapitation. Three brain parts (cerebral cortex, corpus striatum and medulla oblongata), a liver sample and a blood sample were obtained from each rat at the time of euthanasia, and were chilled. Treatment groups were control rats (sham-operated and partially hepatectomized) receiving propylene glycol sampled at 30 min, and treated rats (sham-operated and partially hepatectomized) receiving 1.5 mg/kg parathion at 5 min and 30 min. The samples at 5 min were taken to determine any initial AChE inhibition resulting from this level of parathion. Acetylcholinesterase: AChE activity was assessed by a modification of the Ellman et al. (18) technique, as previously described (19). The concentrations used were: cerebral cortex, i mg/ml; corpus striatum, 0.125 mg/ml; and medulla oblongata, 0.833 mg/ml. Protein was quantified with the Folin phenol reagent, with bovine serum albumin as the standard (20). Aliesterases: Aliesterase activity was assayed in liver and plasma samples as previously described with 4-nitrophenyl valerate as the substrate (9), except that the liver was assayed without centrifugation. The liver and plasma concentrations were 0.025 mg/ml and 1.6 ~I/ml, respectively. Protein was quantified as described above. Statistics: An analysis of variance was performed on each data set using the SAS computer program, with mean separation by the Least Significant Difference method (LSD) or the Student-Newman-Keuls test (SNK). Results Dosages of parathion between 1.2 and 2.4 mg/kg were initially tested. The latter was the lower dosage used in the previously reported ligation experiments (17); this dosage proved to be lethal within 5-10 min in the
1026
Target Organ Bioactivation of Parathion
partially hepatectomized rats. desired effect.
Vol. 48, No. I0, 1991
The dosage used, 1.5 mg/kg, yielded the
As expected, hepatectomy did not affect brain AChE activity (Table I). Also, there was no inhibition of AChE at 5 mln in either the sham or the hepatectomized animals, nor in the sham animals after 30 min. Although there were some statistically significant differences among these groups in the medulla oblongata, they seem to have resulted from inexplicably high sham control values. However, there was a dramatic inhibition of AChE of about 68% in all three brain parts in the hepatectomized animals 30 min after parathion injection.
TABLE I Acetylcholinesterase Activities in Brain Parts from Rats Receiving Intravenous Parathion in Propylene Glycol a
Specific Activity b
Time, min
Cerebral Cortex
Corpus Striatum
Medulla Oblongata
30 30
57.9± 7.1 A 51.3±11.9 A
393±35 A 376±29 A
87.4± 3.2 A 79.6± 4.9 B
Parathion Parathion
5 5
49.9±11.2 A 51.5± 5.3 A
378±59 A 364±43 A
72.0± 2.3 B 74.9± 3.3 B
Parathion Parathion
30 30
49.5± 5.5 A 18.3± 6.8 B
345±18 A 124±52 B
68.5±11.7 B 28.9±10.0 C
Condition
Treatment
Sham Hepatectomy
Control Control
Sham Hepatectomy Sham Hepatectomy
aParathion dosage was 1.5 mg/kg. bSpecific activities are expressed in nmoles/min/mg protein, mear~+S.E.M. All means are the result of 3 or 4 replications. Means within a brain part not followed by the same letter are significantly different by LSD (P < 0.05).
Liver aliesterase activity was also unaffected by hepatectomy (Table II). Liver aliesterases were substantially inhibited (about 46%) at 30 min after parathion injection in the sham-operated animals but were not inhibited in the partially hepatectomized rats. The only significant difference among plasma aliesterase activities was in the sham-operated treated animals at 30 min (Table III). Additionally, when the individual differences between pre-injection and post-injection activities were compared, it can be seen, again, that the only significant depression of aliesterase activity in the plasma was in the sham-operated group at 30 min (92%). Discussion The results of this experiment clearly indicate that the brain is capable of bioactivating parathion at realistic circulating concentrations of the insecticide. Although we have not determined a lethal dosage of parathion
Vol. 48, No.
I0, 1991
Target Organ Bioactivation
of Parathion
1027
TABLE II Hepatic Aliesterase Activities from Male Rats Receiving Intravenous Parathion in Propylene Glycol"
Specific A c t i v l t ~
Condition
Treatment
Sham Hepatectomy
Control Control
Sham Hepatectomy
Parathion Parathion
Sham Hepatectomy
Parathion Parathion
Time
Pre-in~ection
Post-injection
30 30
-891.8±121.1 A
924.1±134.5 A 843.8±114.7 A
5 5
-897.4±111.7
A
934.0±203.7 A 870.1±133.3 A
30 30
-848.6±243.6
A
497.0±152.1 B 849.5±337.4 A
"Parathion dosage was 1.5 mg/kg. bSpecific activities are expressed as nmoles/min/mg protein, mear~+S.E.M. means are the result of 4 replications. Means not followed by the same letter are significantly different by LSD (P < 0.05).
All
TABLE III Plasma Aliesterase Activity from Male Rats Receiving Intravenous Parathion in Propylene Glycol a
Specific A c t i v i t ~
Condition
Treatment
Time
Pre-injection
Post-injection
Sham Hepatectomy
Control Control
Sham Hepatectomy Sham Hepatectomy
Differences =
30 30
208.4±53.7 A 164.5±18.6 A
191.8±13.6 A 152.9±22.5 A
27.8±17.6 A 13.9±11.4 A
Parathion Parathion
5 5
195.6±25.3 A 144.1± 5.9 A
177.6±12.6 A 146.7±12.8 A
17.9±15.1 A 6.8± 4.3 A
Parathion Parathion
30 30
176.0±26.2 A 157.8±31.8 A
12.0± 4.7 B 146.0±22.3 A
163.5±28.7 B 13.9±13.5 A
aParathlon dosage was 1.5 mg/kg. bSpecific activities are expressed as nmoles/min/mg protein, mean!+S.E.M. means are the result of 4 replications. Means not followed by the same letter are significantly different by SNK (P < 0.05).
All
=Difference in specific activities between each rat's post-injection sample to its pre-lnjection sample. Means not followed by the same letter are significantly different by LSD (P < 0.05).
1028
Target Organ Bioactivation of Parathion
Vol. 48, No. I0, 1991
by the i.v. route, an i.p. injection of 15 mg/kg was near lethal in male rats in 60 min (19), and the reported oral 13)50 for male Sherman rats was 13 mg/kg (21). Thus we feel that the dosage used here (1.5 mg/kg) approximated a level which is responsible for acute lethality. The partial hepatectomy procedure allowed a time frame to be employed (30 min) which is the same as the time in which obvious signs of poisoning are observed in the intact animal. This procedure clearly has an advantage over the earlier ligation experiments (17) in that such severe anatomical and physiological disruptions were avoided. Nevertheless, the fact that 30% of the liver remained left a concern that the residual liver could still contribute oxon. However, as evidenced by the fact that liver aliesterase activity was unaffected in the remaining liver 30 min after parathion administration, it appears that hepatic desulfuration activity ceased as a consequence of this surgery. Since the rat hepatic aliesterases are 17 times more sensitive to paraoxon inhibition in vitro than the brain AChE (based on 150 values) (9), if appreciable quantities of oxon had been produced by the liver during this time, the aliesterase activities should have reflected the production. Thus, it can be concluded that the residual liver did not contribute oxon to the system. The results obtained with the sham-operated animals at the same parathion dosage, i.e., massive inhibition of hepatic and plasma aliesterases and no inhibition of brain ACHE, clearly illustrate the ability of the liver and the blood to sequester the phosphorothionate and/or its oxon. While extra-hepatic tissues besides the brain could also have been a source of oxon in the hepatectomized rats, this was not the case because the plasma aliesterases were not inhibited 30 min after parathion administration. Because the brain AChE activities obtained in the hepatectomized rats 5 min after parathion administration should have reflected inhibition resulting from the parathion sample itself, it is clear that paraoxon was produced during the 30 minute experimental period. It is unknown whether neurons or glial cells were responsible for this. P-450 activities have been detected in brain microvessels (22); hence, it is also possible that the brain's capillaries were responsible for oxon generation. In conclusion, these experiments have demonstrated that the brain is capable of generating sufficient quantities of paraoxon to inhibit AChE substantially at a realistic concentration of circulating parathion in a realistic period of time. Thus, target organ activation of parathion has been demonstrated in v i v o at sufficient levels to contribute to toxicity. Acknowledgments This research was supported by grant ES04394 from the National Institutes of Health, and by Research Career Development Award ESO0190 to J.E.C. The authors appreciate the excellent technical assistance of Jeanne Taylor, the animal care rendered by Michael Bassett, and the guidance for surgical procedures provided by John E. Harkness, D.V.M.
References i.
2. 3.
R. NEAL, Reviews in Biochemical Toxicology, Vol. 2, E. Hodgson, J.R. Bend, and R.M. Philpot (eds.), 131-171, Elsevier North Holland, New York (1980). C.S. FORSYTH AND J.E. CHAMBERS, Biochem. Pharmacol. 38 1597-1603 (1989). J.E. CHAMBERS, C.S. FORSYTH and H.W. CHAMBERS, Intermediary Xenobiotic
Vol. 48, No. I0, 1991
4.
5. 6. 7. 8. 9. i0. Ii. 12. 13. 14. 15 16 17. 18. 19. 20. 21. 22.
Target Organ Bioactivation of Parathion
1029
Metabolism: Methodolozv. Mechanisms and Significance, J. Caldwell, D.H. Hutson, and G.D. Paulson (eds), 99-115, Taylor and Francis, Baslngstoke, U.K. (1988). S.D. MURPHY, Casarett and Doull's Toxicology, C.D. Klaassen, M.O. Amdur, and J. Doull (eds), 519-581, Macmillan, New York (1986). R.R. LAUWERYS and S.D. MURPHY, Toxicol. Appl. Fharmacol. 14 348-357 (1969). F. FONNUM and S.H. STERRI, Fundam. AppI. Toxicol. ! 143-147 (1981). J.G. CLEMENT, Fundam. AppI. Toxicol. ~ S96 (1984). D.M. MAXWELL, K.M. BRECHT, D.E. LENZ and B.L. O'NEILL, J. Pharmacol. Exp. Ther. 246 986-991 (1988). H. CHAMBERS, B. BROWN and J.E. CHAMBERS, Pestic. Biochem. Physiol. 36 308-315 (1990). J.M. BECKER and T. NAKATSUGAWA, Pestic. Biochem. Physiol. 36 83-98 (1990). M.K. QATO and M.D. MAINES, Biochem. Biophys. Res. Comm. 128 18-24 (1985). M. MESNIL, B. TESTA and P. JENNER, Biochem. Pharmacol. 3-4 435-436 (1985). B. WALTHER, J.F. GHERSI-EGEA, A. MINN and G. SIEST, Brain Res. 375 388344 (1986). B. WALTHER, J.-F. GHERSI-EGEA, A. MINN and G. SIEST, Arch. Biochem. Biophys. 254 592-596 (1987). B. WALTHER, J.-F. GHERSI-EGEA, Z. JAYYOSI, A. MINN and G. SIEST, Neurosci. Lett. 76 58-62 (1987). J.E. CHAMBERS and H.W. CHAMBERS, J. Biochem. Toxicol. ~ 201-203 (1989). J.E. CHAMBERS, J.R. MUNSON, and H.W. CHAMBERS, Biochem. Biophys. Res. Comm. 165 327-333 (1989). G.L. ELI2[AN, K.D. COURTNEY, V. ANDRES, JR. and R.M. FEATHERSTONE, Biochem. Fharmacol. ! 88-95 (1961). H.W. CHAMBERS and J.E. CHAMBERS, Festic. Biochem. Physiol. 33 125-131 (1989). O.H. LOWRY, N.J. ROSEBROUGH, A.L. FARR and R.J. RANDALL, J. Biol. Chem. 193 265-275 (1951). T.B. GAINES, Toxicol. AppI. Pharmacol. 2 88-89 (1960). J.F. GHERSI-EGEA, A. MINN and G. SIEST, Life Sci. 4_/2 2515-2523 (1988).
Pergamon Press
TARGET SITE BIOACTIVATION OF THE NEUROTOXIC ORGANOPHOSPHORUS INSECTICIDE PARATHION IN PARTIALLY HEPATECTOMIZED RATS Janice E. Chambers I, Howard W. Chambers and John E. Snawder 2 Depts. of Biological Sciences and Entomology, Mississippi State University, Mississippi State, Mississippi (Received in final form January 7, 1991) Summary Target organ bioactivation of phosphorothionate insecticides to their potent anticholinesterase oxon metabolites (for example, parathion to paraoxon) may be extremely important in toxicity because liver and blood provide so much potential protection by a variety of mechanisms, such as the aliesterases which serve as alternate phosphorylation sites. To determine whether the brain can produce sufficient oxon in vivo to contribute to toxicity, male rats were partially hepatectomized and injected i.v. with 1.5 mg/kg parathion. After 30 minutes, brain AChE was inhibited 68% whereas liver and plasma aliesterases were unaffected. Because aliesterases are far more sensitive to paraoxon inhibition than is brain ACHE, these results indicate that neither the liver nor extra-hepatic tissues were contributing oxon into the blood stream. Thus target site activation of parathion occurred in vivo at sufficient levels to contribute substantially to toxicity. Many xenobiotics require bioactivation to display toxicity because toxic effects are mediated by covalent binding between a reactive metabolite and the target molecule. The liver is large and has very high activities of the xenobiotic metabolizing enzymes which are responsible for bioactivation reactions, usually cytochrome P-450-dependent monooxygenases (P-450). In the case of non-hepatotoxic xenobiotics, however, these metabolites will need to react with target molecules distant from the liver, thus requiring transport. It seems unlikely that appreciable quantities of hepatically-generated reactive metabolite would be able to reach the target tissue intact if very many alternative reactive sites exist in either the liver or the blood stream. In such cases, it may be a criterion for toxicity that bioactivation of the xenobiotic occur within the target tissue so that formation of very labile metabolites would occur in close proximity to the target molecules. The above scenario is quite likely for phosphorothionate insecticides, the most common chemical form of organophosphorus insecticides. The phosphorothionate is metabolized by P-450 to a potent anticholinesterase, the oxon or phosphate; for example, parathion is converted to paraoxon (i). The bioactivation increases the anticholinesterase potency by about three orders of magnitude (2). The phosphorylation of the serine hydroxyl at the active site of acetylcholinesterase (ACHE) within the nervous system is currently IAddress correspondence to: Department of Biological Sciences, Mississippi State University, P.O. Drawer GY, Mississippi State, MS 39762, USA. ZPresent address: National Center for Toxicological Research, Jefferson, AR. 0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc
1024
Target Organ Bioactlvation of Parathion
Vol. 48, No. I0, 1991
accepted as the mechanism of acute toxicity (4). However, the oxons react not only with AChE but also with other serlne esterases and with serine proteases. Phosphorylation of these non-target serine esterases/proteases serves to stoichiometrically destroy the oxon. Enzymes that can participate in this non-catalytlc detoxication process are hepatic aliesterases (carboxylesterases), plasma allesterases and butyrylcholinesterase, erythrocyte ACHE, and serine proteases in a variety of tissues. The high enzyme activities, implying high concentrations, of aliesterases in liver and plasma suggest that they may be particularly important in protecting the organism from intoxication by organophosphates. Selective inhibition of the allesterases in vivo resulted in enhanced toxicity of the organephosphate paraoxon (5). The allesterases have been implicated as alternate phosphorylation sites in poisoning by the organophosphate nerve agents (6-8). Because the hepatic aliesterases are about an order of magnitude more sensitive than brain ACHE, they are particularly likely to sequester oxon as it is generated within the liver (9). In fact, the liver effectively retains most of the parathion perfused into it with little paraoxon observed escaping from the perfused liver (i0), indicating that the liver is a very effective trap for any paraoxon it generates. Thus, only low quantities of oxon should be expected to escape the liver unless the dose were so high as to overwhelm hepatic protective mechanisms. What little escapes could then be sequestered by the protective factors in the plasma. Target organ activation, then, may be responsible for generating most of the oxon which inhibits nervous system ACHE. The brain possesses a variety of cytochrome P-450-dependent monooxygenase activities (11-15). Both the microsomal and mitochondrial fractions of brain possess desulfuration activities for six phosphorothionates (2,3,16). In these studies there was a very good correlation between in vitro brain desulfuration activity and acute toxicity level, whereas there was no correlation between liver desulfuration activity and toxicity. This relationship suggested that brain desulfuration activity may have generated the oxon responsible for toxicity and that the level of brain activation was an important determinant of acute toxicity level. Nevertheless, the brain desulfuration activity is extremely low compared with the liver, so the question remains as to whether the brain could actually generate significant amounts of oxon. Experiments were conducted in the rat following ligation of the descending aorta, thus removing the liver from the circulation of the brain. In this protocol with an extremely high dose of parathion and a 15 minute time frame, a significant amount of brain AChE inhibition occurred (17). The short time frame was necessitated by the large amount of the body (about 60%) which had been removed from the circulation, and the extremely high dose was necessitated by the short time frame. This paper describes a protocol involving partial hepatectomy which allowed a lower dose and longer time frame to be employed to mimic more closely the conditions of a realistic phosphorothionate intoxication. As with the previous ligation experiments (17), brain AChE inhibition was assessed following parathion administration. Also assessed was the inhibition of hepatic and plasma aliesterases, which are an order of magnitude more sensitive to paraoxon than brain ACHE, and therefore provide a highly sensitive indicator of the presence of paraoxon. Materials and Methods Animals: Male Sprague-Dawley derived rats [CrI:CD(SD)BR] from an original Charles River stock weighing about 400-450 g were used. Before use, they were housed in a temperature-controlled animal room with a 12:12 hr light cycle and with Purina laboratory rodent food and tap water freely available.
Vol. 48, No. I0, 1991
Target Organ Bioactivation
of Parathion
1025
Chemicals: Parathion was a gift of Monsanto Chemical Company (St. Louis, MO) and was purified by recrystalllzatlon from methanol. Chemicals for enzyme assays were obtained from Sigma Chemical Company (St. Louis, MO). Surgical procedure and treatment: Rats were anesthetized with methoxyflurane and were maintained throughout surgery under anesthesia. All surgical procedures were conducted in a fume hood. The animal's abdomen was shaved and an incision was made to expose the liver. All lobes of the liver except the right lateral lobe and the caudal lobe, through which the posterior vena cava courses, were removed following llgation of the lobe at its base. Removal of these two remaining lobes would have required much more prolonged and disruptive surgery than was employed. This procedure removed about 70% (6872%) of the liver mass. The liver removed was chilled for use in a preinjection allesterase determination. The incision in the abdominal muscle wall was closed with sutures and the skin was closed with wound clips. Sham operations were also performed. Prior to recovery from anesthesia, treatment with 1.5 mg/kg parathion in propylene glycol was administered as an i.v. injection into the tail vein in a volume of 0.5 ml/kg. Propylene glycol in the same volume was administered to controls. Prior to injection the end of the tail was cut to obtain a blood sample which was collected into a 0.4 ml microcentrifuge tube containing a few mg of solid EDTA as an anticoagulant. The blood was gently mixed with the EDTA and the sample was chilled. Plasma was obtained by centrifugation at room temperature in an Eppendorf microcentrifuge at 17,000 g for 5 min. Each animal was allowed to recover from anesthesia. At the appropriate time each was euthanitized by decapitation. Three brain parts (cerebral cortex, corpus striatum and medulla oblongata), a liver sample and a blood sample were obtained from each rat at the time of euthanasia, and were chilled. Treatment groups were control rats (sham-operated and partially hepatectomized) receiving propylene glycol sampled at 30 min, and treated rats (sham-operated and partially hepatectomized) receiving 1.5 mg/kg parathion at 5 min and 30 min. The samples at 5 min were taken to determine any initial AChE inhibition resulting from this level of parathion. Acetylcholinesterase: AChE activity was assessed by a modification of the Ellman et al. (18) technique, as previously described (19). The concentrations used were: cerebral cortex, i mg/ml; corpus striatum, 0.125 mg/ml; and medulla oblongata, 0.833 mg/ml. Protein was quantified with the Folin phenol reagent, with bovine serum albumin as the standard (20). Aliesterases: Aliesterase activity was assayed in liver and plasma samples as previously described with 4-nitrophenyl valerate as the substrate (9), except that the liver was assayed without centrifugation. The liver and plasma concentrations were 0.025 mg/ml and 1.6 ~I/ml, respectively. Protein was quantified as described above. Statistics: An analysis of variance was performed on each data set using the SAS computer program, with mean separation by the Least Significant Difference method (LSD) or the Student-Newman-Keuls test (SNK). Results Dosages of parathion between 1.2 and 2.4 mg/kg were initially tested. The latter was the lower dosage used in the previously reported ligation experiments (17); this dosage proved to be lethal within 5-10 min in the
1026
Target Organ Bioactivation of Parathion
partially hepatectomized rats. desired effect.
Vol. 48, No. I0, 1991
The dosage used, 1.5 mg/kg, yielded the
As expected, hepatectomy did not affect brain AChE activity (Table I). Also, there was no inhibition of AChE at 5 mln in either the sham or the hepatectomized animals, nor in the sham animals after 30 min. Although there were some statistically significant differences among these groups in the medulla oblongata, they seem to have resulted from inexplicably high sham control values. However, there was a dramatic inhibition of AChE of about 68% in all three brain parts in the hepatectomized animals 30 min after parathion injection.
TABLE I Acetylcholinesterase Activities in Brain Parts from Rats Receiving Intravenous Parathion in Propylene Glycol a
Specific Activity b
Time, min
Cerebral Cortex
Corpus Striatum
Medulla Oblongata
30 30
57.9± 7.1 A 51.3±11.9 A
393±35 A 376±29 A
87.4± 3.2 A 79.6± 4.9 B
Parathion Parathion
5 5
49.9±11.2 A 51.5± 5.3 A
378±59 A 364±43 A
72.0± 2.3 B 74.9± 3.3 B
Parathion Parathion
30 30
49.5± 5.5 A 18.3± 6.8 B
345±18 A 124±52 B
68.5±11.7 B 28.9±10.0 C
Condition
Treatment
Sham Hepatectomy
Control Control
Sham Hepatectomy Sham Hepatectomy
aParathion dosage was 1.5 mg/kg. bSpecific activities are expressed in nmoles/min/mg protein, mear~+S.E.M. All means are the result of 3 or 4 replications. Means within a brain part not followed by the same letter are significantly different by LSD (P < 0.05).
Liver aliesterase activity was also unaffected by hepatectomy (Table II). Liver aliesterases were substantially inhibited (about 46%) at 30 min after parathion injection in the sham-operated animals but were not inhibited in the partially hepatectomized rats. The only significant difference among plasma aliesterase activities was in the sham-operated treated animals at 30 min (Table III). Additionally, when the individual differences between pre-injection and post-injection activities were compared, it can be seen, again, that the only significant depression of aliesterase activity in the plasma was in the sham-operated group at 30 min (92%). Discussion The results of this experiment clearly indicate that the brain is capable of bioactivating parathion at realistic circulating concentrations of the insecticide. Although we have not determined a lethal dosage of parathion
Vol. 48, No.
I0, 1991
Target Organ Bioactivation
of Parathion
1027
TABLE II Hepatic Aliesterase Activities from Male Rats Receiving Intravenous Parathion in Propylene Glycol"
Specific A c t i v l t ~
Condition
Treatment
Sham Hepatectomy
Control Control
Sham Hepatectomy
Parathion Parathion
Sham Hepatectomy
Parathion Parathion
Time
Pre-in~ection
Post-injection
30 30
-891.8±121.1 A
924.1±134.5 A 843.8±114.7 A
5 5
-897.4±111.7
A
934.0±203.7 A 870.1±133.3 A
30 30
-848.6±243.6
A
497.0±152.1 B 849.5±337.4 A
"Parathion dosage was 1.5 mg/kg. bSpecific activities are expressed as nmoles/min/mg protein, mear~+S.E.M. means are the result of 4 replications. Means not followed by the same letter are significantly different by LSD (P < 0.05).
All
TABLE III Plasma Aliesterase Activity from Male Rats Receiving Intravenous Parathion in Propylene Glycol a
Specific A c t i v i t ~
Condition
Treatment
Time
Pre-injection
Post-injection
Sham Hepatectomy
Control Control
Sham Hepatectomy Sham Hepatectomy
Differences =
30 30
208.4±53.7 A 164.5±18.6 A
191.8±13.6 A 152.9±22.5 A
27.8±17.6 A 13.9±11.4 A
Parathion Parathion
5 5
195.6±25.3 A 144.1± 5.9 A
177.6±12.6 A 146.7±12.8 A
17.9±15.1 A 6.8± 4.3 A
Parathion Parathion
30 30
176.0±26.2 A 157.8±31.8 A
12.0± 4.7 B 146.0±22.3 A
163.5±28.7 B 13.9±13.5 A
aParathlon dosage was 1.5 mg/kg. bSpecific activities are expressed as nmoles/min/mg protein, mean!+S.E.M. means are the result of 4 replications. Means not followed by the same letter are significantly different by SNK (P < 0.05).
All
=Difference in specific activities between each rat's post-injection sample to its pre-lnjection sample. Means not followed by the same letter are significantly different by LSD (P < 0.05).
1028
Target Organ Bioactivation of Parathion
Vol. 48, No. I0, 1991
by the i.v. route, an i.p. injection of 15 mg/kg was near lethal in male rats in 60 min (19), and the reported oral 13)50 for male Sherman rats was 13 mg/kg (21). Thus we feel that the dosage used here (1.5 mg/kg) approximated a level which is responsible for acute lethality. The partial hepatectomy procedure allowed a time frame to be employed (30 min) which is the same as the time in which obvious signs of poisoning are observed in the intact animal. This procedure clearly has an advantage over the earlier ligation experiments (17) in that such severe anatomical and physiological disruptions were avoided. Nevertheless, the fact that 30% of the liver remained left a concern that the residual liver could still contribute oxon. However, as evidenced by the fact that liver aliesterase activity was unaffected in the remaining liver 30 min after parathion administration, it appears that hepatic desulfuration activity ceased as a consequence of this surgery. Since the rat hepatic aliesterases are 17 times more sensitive to paraoxon inhibition in vitro than the brain AChE (based on 150 values) (9), if appreciable quantities of oxon had been produced by the liver during this time, the aliesterase activities should have reflected the production. Thus, it can be concluded that the residual liver did not contribute oxon to the system. The results obtained with the sham-operated animals at the same parathion dosage, i.e., massive inhibition of hepatic and plasma aliesterases and no inhibition of brain ACHE, clearly illustrate the ability of the liver and the blood to sequester the phosphorothionate and/or its oxon. While extra-hepatic tissues besides the brain could also have been a source of oxon in the hepatectomized rats, this was not the case because the plasma aliesterases were not inhibited 30 min after parathion administration. Because the brain AChE activities obtained in the hepatectomized rats 5 min after parathion administration should have reflected inhibition resulting from the parathion sample itself, it is clear that paraoxon was produced during the 30 minute experimental period. It is unknown whether neurons or glial cells were responsible for this. P-450 activities have been detected in brain microvessels (22); hence, it is also possible that the brain's capillaries were responsible for oxon generation. In conclusion, these experiments have demonstrated that the brain is capable of generating sufficient quantities of paraoxon to inhibit AChE substantially at a realistic concentration of circulating parathion in a realistic period of time. Thus, target organ activation of parathion has been demonstrated in v i v o at sufficient levels to contribute to toxicity. Acknowledgments This research was supported by grant ES04394 from the National Institutes of Health, and by Research Career Development Award ESO0190 to J.E.C. The authors appreciate the excellent technical assistance of Jeanne Taylor, the animal care rendered by Michael Bassett, and the guidance for surgical procedures provided by John E. Harkness, D.V.M.
References i.
2. 3.
R. NEAL, Reviews in Biochemical Toxicology, Vol. 2, E. Hodgson, J.R. Bend, and R.M. Philpot (eds.), 131-171, Elsevier North Holland, New York (1980). C.S. FORSYTH AND J.E. CHAMBERS, Biochem. Pharmacol. 38 1597-1603 (1989). J.E. CHAMBERS, C.S. FORSYTH and H.W. CHAMBERS, Intermediary Xenobiotic
Vol. 48, No. I0, 1991
4.
5. 6. 7. 8. 9. i0. Ii. 12. 13. 14. 15 16 17. 18. 19. 20. 21. 22.
Target Organ Bioactivation of Parathion
1029
Metabolism: Methodolozv. Mechanisms and Significance, J. Caldwell, D.H. Hutson, and G.D. Paulson (eds), 99-115, Taylor and Francis, Baslngstoke, U.K. (1988). S.D. MURPHY, Casarett and Doull's Toxicology, C.D. Klaassen, M.O. Amdur, and J. Doull (eds), 519-581, Macmillan, New York (1986). R.R. LAUWERYS and S.D. MURPHY, Toxicol. Appl. Fharmacol. 14 348-357 (1969). F. FONNUM and S.H. STERRI, Fundam. AppI. Toxicol. ! 143-147 (1981). J.G. CLEMENT, Fundam. AppI. Toxicol. ~ S96 (1984). D.M. MAXWELL, K.M. BRECHT, D.E. LENZ and B.L. O'NEILL, J. Pharmacol. Exp. Ther. 246 986-991 (1988). H. CHAMBERS, B. BROWN and J.E. CHAMBERS, Pestic. Biochem. Physiol. 36 308-315 (1990). J.M. BECKER and T. NAKATSUGAWA, Pestic. Biochem. Physiol. 36 83-98 (1990). M.K. QATO and M.D. MAINES, Biochem. Biophys. Res. Comm. 128 18-24 (1985). M. MESNIL, B. TESTA and P. JENNER, Biochem. Pharmacol. 3-4 435-436 (1985). B. WALTHER, J.F. GHERSI-EGEA, A. MINN and G. SIEST, Brain Res. 375 388344 (1986). B. WALTHER, J.-F. GHERSI-EGEA, A. MINN and G. SIEST, Arch. Biochem. Biophys. 254 592-596 (1987). B. WALTHER, J.-F. GHERSI-EGEA, Z. JAYYOSI, A. MINN and G. SIEST, Neurosci. Lett. 76 58-62 (1987). J.E. CHAMBERS and H.W. CHAMBERS, J. Biochem. Toxicol. ~ 201-203 (1989). J.E. CHAMBERS, J.R. MUNSON, and H.W. CHAMBERS, Biochem. Biophys. Res. Comm. 165 327-333 (1989). G.L. ELI2[AN, K.D. COURTNEY, V. ANDRES, JR. and R.M. FEATHERSTONE, Biochem. Fharmacol. ! 88-95 (1961). H.W. CHAMBERS and J.E. CHAMBERS, Festic. Biochem. Physiol. 33 125-131 (1989). O.H. LOWRY, N.J. ROSEBROUGH, A.L. FARR and R.J. RANDALL, J. Biol. Chem. 193 265-275 (1951). T.B. GAINES, Toxicol. AppI. Pharmacol. 2 88-89 (1960). J.F. GHERSI-EGEA, A. MINN and G. SIEST, Life Sci. 4_/2 2515-2523 (1988).