TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
SHORT Comparative
Toxicity Neonatal
32,443-446
(1975)
COMMUNICATION of Some Selected and Adult Rats’
Pesticides
in
ComparativeToxicity of SomeSelectedPesticidesin Neonatal and Adult Rats. HARBISON, R. D. (1974). Toxicol. Appl. Pharmcol. 32, 443-446. Toxicity of two groupsof pesticideswasstudiedin adult and newbornrats. The chlorinated group of pesticides,heptachlor, chlordane, and DDT arelesstoxic in the newbornwhencomparedto the adult, but phenobarbital pretreatment potentiates their toxicity in the newborn. The organophosphorusinsecticide,parathion, is more toxic in the newbornwhen compared to adult, but phenobarbital pretreatment antagonizedthe toxicity of this cholinesteraseinhibitor. Chlorinated pesticidesare generally lesstoxic in the newborn, while the pesticidesof the cholinesteraseinhibitor group are more toxic. Pesticides can be bioactivated or detoxified in mammalian systems. The capacity for these processeswould be expected to vary with age development. Also, the capacity of these metabolic processeswould be expected to be altered by phenobarbital pretreatment. The purposes of this study were to: (1) determine the toxicity of various pesticides in the newborn and adult (the pesticides selected include those which are either bioactivated, detoxified or both), and (2) determine the effect of phenobarbital pretreatment on the toxicity of the selectedgroup of pesticides. METHODS Neonatal Sprague-Dawley rats (Harlan Industries, Cumberland, Indiana) 2-5 days postpartum and adult rats 120-150 g were used in this study. Animals were injected intraperitoneally and lethality data gathered for the following 7 consecutive days. Analytical grade dichlorodiphenyltrichlorethane (DDT), chlordane, heptachlor, and parathion were dissolved in corn oil shortly before intraperitoneal injection. Phenobarbital sodium 40 mg/kg was administered intraperitoneally once a day for 3 days prior to pesticide administration. The LD50 and potency ratios were calculated by the method of probits described by Finney (I 947). RESULTS AND DISCUSSION DDT is dehydrochlorinated to DDE, reductively dechlorinated to DDD and oxidatively metabolized to DDA; all metabolites are excreted in the urine. In rats, the signsof DDT toxicity are correlated with DDT concentrations in the brain, regardless of whether DDT is administered in a single large dose or in multiple smaller doses ’ Thisresearch wassupportedin part by ES-00782andES-00267. Copyright 0 1975 by Academic Press, Inc. All rights ofreproduction in any form reserved. Printed in Great Britain
443
SHORT COMMUNICATION
444
(Dale et al., 1963; Hayes and Dale, 1964). Henderson and Woolley (1970) and others have suggested that DDT is detoxified to DDE and DDA. Since DDT is a compound which is detoxified, it would be expected to be more toxic in the newborn because of reduced metabolic capability. Comparative lethality data for DDT for adult, newborn, and phenobarbital-pretreated newborn are shown in Table 1. The newborn is less sensitive to the toxic effects of DDT than the adults. Phenobarbital pretreatment increases the sensitivity of the newborn to DDT toxicity. TABLE
1
COMPARATIVE LD50 VALUES AND CONFIDENCE LIMITS OF SEVERAL PESTICIDES' Adult
Pesticide DDT
Chlordane Heptachlor Parathion
(male) 225 343
(482-84) (451-258)
Newborn (mixed sex) 2356
(3327-1565)
71 (103-41)
1121 (1289-984) 531 (102&311)
8.8 (10.9-7.0)
1.8 (2.0-l .6)
Newborn + PB 1345 539
(1681-857) (666-428)
133 (179-86) 4.8 (5.7-4.1)
Potency ratios and confidence limits Pesticide
Newborn vs adult
DDT Chlordane Heptachlor
10.5 (27.94.5)
Pesticide
Adult vs newborn
Parathion
3.3 (4.5-2.4) 7.5 (16.942)
4.8 (6.1-3.8)
Newborn + PB vs adult Newborn vs newborn + PB 5.9 (6.3-2.6) 1.6 (2.2-1.1) 1.8 (3.4-1.2)
1.8 (2.8-1.2) 2.1 (2.7-1.6) 4.0 (7.9-2.4)
Adult vs newborn + PB Newborn + PB vs newborn 1.8 (2.4-l .4)
2.6 (3.2-2.2)
n Newborn rats 2-5 days postpartum and adult rats 120-150 g were used. Groups of lo-20 animals were used in each experimental group. Pesticides were administered ip in corn oil; phenobarbital (PB) was administered ip.
Metabolism of chlordane is considered to be a detoxification mechanism by direct replacement of chlorine by a hydroxyl group to form chlorohydrin and a diol (Wilkinson, 1968). The newborn is less sensitive to the toxic effects of chlordane than the adult (Table 1). Phenobarbital pretreatment increases the sensitivity of the newborn to chlordane toxicity. Epoxidation of unsaturated cyclodienes such as heptachlor has been shown to result from the activity of a typical microsomal enzyme requiring NADPH and oxygen (Brooks, 1965). Epoxidation is regarded as an activating step rather than detoxication. Comparative lethality data for heptachlor between adult, newborn, and phenobarbital pretreated newborn are shown in Table 1. The newborn is less sensitive to the toxic effects of heptachlor. Phenobarbital pretreatment increases the sensitivity of the newborn to heptachlor toxicity. Thus, the ability to metabolize and bioactivate heptachlor correlates with its toxicity in the newborn. The next pesticide to be considered is parathion. The microsomal enzymes cannot only oxidize parathion to the active cholinesterase inhibitor, paraoxon, but can also
SHORT
COMMUNICATION
445
detoxify parathion to p-nitrophenol and diethyl phosphorothionate. Also, hydrolysis of the phosphorous of paraoxon can yield diethyl phosphate and p-nitrophenol. Since the newborn has low microsomal mixed function oxidase activity, parathion should be less toxic in the newborn. Comparative lethality data for parathion between adult, newborn, and phenobarbital-pretreated newborn are shown in Table 1. Paradoxically, the newborn is more sensitive to the toxic effects of parathion. Phenobarbital pretreatment decreases the sensitivity of the newborn to parathion toxicity. Thus, inducing the metabolism of parathion in the newborn appears to enhance detoxification rather than bioactivation. Often, pesticides are more toxic in the newborn than the adult (Lu et al., 1965). However, of the four pesticides studied, three of these, DDT, chlordane, and heptachlor were less toxic in the newborn. DuBois and Puchala (1961), Murphy and DuBois (1958), and Brodeur and DuBois (1963; 1967) reported that most organophosphate insecticides are more toxic to weanling rats than adults. Similarly, the results presented here demonstrate parathion is more toxic in the newborn. Toxicity differences may result from differences in cholinesterase activities between the two age groups. Protection against the toxicity of various insecticides of the organophosphorous series has been reported following pretreatment with drugs known to induce hepatic microsomal enzyme activity (Ball et al., 1954; Welch and Coon, 1964; DuBois and Kinoshita, 1966). More recently, Alary and Brodeur (1969) reported phenobarbital protected against parathion toxicity in adult female rats. The data presented here demonstrates phenobarbital pretreatment potentiates the toxicity of DDT, chlordane, and heptachlor in the newborn. Phenobarbital may stimulate production of a metabolite inherently more toxic than the parent compound in newborn rats. Phenobarbital pretreatment, however, did protect against parathion toxicity. Alary and Brodeur (1969) reported phenobarbital stimulates the direct degradation of parathion to diethyl phosphorothioic acid and p-nitrophenol. Hydrolysis of paraoxon by the newborn is probably deficient. REFERENCES ALARY, J. G. AND BRODEUR, J. (1969). Studies on the mechanism of phenobarbital-induced protection against parathion in adult female rats. J. Pharmacol. Exp. Ther. 169, 159-167. BALL, W. J., SINCLAIR, J. W., CREVIEW, M. AND KAY, K. (1954). Modification of parathion’s toxicity for rats by pretreatment with chlorinated hydrocarbon insecticides. Can. J. Biochem. Physiol. 32,4%445. BRODEUR, J. AND DUBOIS,K. P. (1963). Comparison of acute toxicity of cholinesterase insecticides to weanling and adult male rats. Proc. Sot. Exp. Biol. Med. 114,509-511. BRODEUR, J. AND DUBOIS, K. P. (1967). Studies on factors influencing the acute toxicity of malathion and malaoxon in rats. Can. J. Physiol. Pharmacol. 45, 621-63 1. BROOKS, G. T. (1965). Structure activity relationship among insecticidal compounds derived from chlordane. Nature (London) 205, 1031-1032. DALE, W. E., GAINES, T. B., HAYES, W. J. AND PEARCE, G. W. (1963). Poisoning by DDT. Relation between clinical signs and concentration in rat brain. Science 142, 1474-1476. DuBors, K. P. AND PUCHALA, E. (1961). Studies on the sex difference in toxicity of a cholinergic phosphorothioate. Proc. Sot. Exp. Biol. Med. 107, 908-911. DuBors, K. P. AND KINOSHITA, F. (1966). Stimulation of detoxification of O-ethyl 0-i4nitrophenyl-phenyl-phosphonothiate (EPN) by nikethamide and phenobarbital. Proc. Sot.
Exp. Biol. Med. 121, 59-62.
446
SHORT COMMUNICATION
FINNEY, D. J. (1947). Probit Analysis. CambridgeUniversity Press. HAYES,W. J. ANDDALE,W. E. (1964). Concentration of DDT in brain and other tissuesin relation to symptomatology. Toxicol. AppE. Pharmacol. 6, 349. HENDERSON, G. L. ANDWOOLLEY,D. A. (1970). Ontogenesisof drug-inducedtremor in the rat. J. Pharmacol. Exp. Thu. 175, 113-120. Lu, F. C., JESSUP, D. C. ANDLAVALLEE,A. (1965).Toxicity of pesticidesin young versusadult rats. Fd. Cosmet. Toxicol. 3, 591-596. MURPHY,S. D. ANDDUBOIS,K. P. (1958).The influenceof various factors on the enzymatic conversion of organic thiophosphatesto anticholinesteraseagents.J. Pharmacol. Exp. Ther. 124, 194202. WELCH,R. M. ANDCOON,J. M. (1964).Studieson the effect of chlorcyclizine and other drugs on the toxicity of several organophosphateanticholinesterases. Pharmacol. Exp. Ther. 143,192-198.
WILKINSON,C. F. (1968). Detoxification of pesticidesand the mechanismof synergism.In The Enzymatic Oxidation of Toxicants (Ernest Hodgson, ed.). North Carolina State University Press,Raleigh,North Carolina. RAYMONDD. HARBISON Center in Toxicology Vanderbilt Medical Center Nashville, Tennessee 37232 Received September 20,1974;
accepted November 25,1974
AND
APPLIED
PHARMACOLOGY
SHORT Comparative
Toxicity Neonatal
32,443-446
(1975)
COMMUNICATION of Some Selected and Adult Rats’
Pesticides
in
ComparativeToxicity of SomeSelectedPesticidesin Neonatal and Adult Rats. HARBISON, R. D. (1974). Toxicol. Appl. Pharmcol. 32, 443-446. Toxicity of two groupsof pesticideswasstudiedin adult and newbornrats. The chlorinated group of pesticides,heptachlor, chlordane, and DDT arelesstoxic in the newbornwhencomparedto the adult, but phenobarbital pretreatment potentiates their toxicity in the newborn. The organophosphorusinsecticide,parathion, is more toxic in the newbornwhen compared to adult, but phenobarbital pretreatment antagonizedthe toxicity of this cholinesteraseinhibitor. Chlorinated pesticidesare generally lesstoxic in the newborn, while the pesticidesof the cholinesteraseinhibitor group are more toxic. Pesticides can be bioactivated or detoxified in mammalian systems. The capacity for these processeswould be expected to vary with age development. Also, the capacity of these metabolic processeswould be expected to be altered by phenobarbital pretreatment. The purposes of this study were to: (1) determine the toxicity of various pesticides in the newborn and adult (the pesticides selected include those which are either bioactivated, detoxified or both), and (2) determine the effect of phenobarbital pretreatment on the toxicity of the selectedgroup of pesticides. METHODS Neonatal Sprague-Dawley rats (Harlan Industries, Cumberland, Indiana) 2-5 days postpartum and adult rats 120-150 g were used in this study. Animals were injected intraperitoneally and lethality data gathered for the following 7 consecutive days. Analytical grade dichlorodiphenyltrichlorethane (DDT), chlordane, heptachlor, and parathion were dissolved in corn oil shortly before intraperitoneal injection. Phenobarbital sodium 40 mg/kg was administered intraperitoneally once a day for 3 days prior to pesticide administration. The LD50 and potency ratios were calculated by the method of probits described by Finney (I 947). RESULTS AND DISCUSSION DDT is dehydrochlorinated to DDE, reductively dechlorinated to DDD and oxidatively metabolized to DDA; all metabolites are excreted in the urine. In rats, the signsof DDT toxicity are correlated with DDT concentrations in the brain, regardless of whether DDT is administered in a single large dose or in multiple smaller doses ’ Thisresearch wassupportedin part by ES-00782andES-00267. Copyright 0 1975 by Academic Press, Inc. All rights ofreproduction in any form reserved. Printed in Great Britain
443
SHORT COMMUNICATION
444
(Dale et al., 1963; Hayes and Dale, 1964). Henderson and Woolley (1970) and others have suggested that DDT is detoxified to DDE and DDA. Since DDT is a compound which is detoxified, it would be expected to be more toxic in the newborn because of reduced metabolic capability. Comparative lethality data for DDT for adult, newborn, and phenobarbital-pretreated newborn are shown in Table 1. The newborn is less sensitive to the toxic effects of DDT than the adults. Phenobarbital pretreatment increases the sensitivity of the newborn to DDT toxicity. TABLE
1
COMPARATIVE LD50 VALUES AND CONFIDENCE LIMITS OF SEVERAL PESTICIDES' Adult
Pesticide DDT
Chlordane Heptachlor Parathion
(male) 225 343
(482-84) (451-258)
Newborn (mixed sex) 2356
(3327-1565)
71 (103-41)
1121 (1289-984) 531 (102&311)
8.8 (10.9-7.0)
1.8 (2.0-l .6)
Newborn + PB 1345 539
(1681-857) (666-428)
133 (179-86) 4.8 (5.7-4.1)
Potency ratios and confidence limits Pesticide
Newborn vs adult
DDT Chlordane Heptachlor
10.5 (27.94.5)
Pesticide
Adult vs newborn
Parathion
3.3 (4.5-2.4) 7.5 (16.942)
4.8 (6.1-3.8)
Newborn + PB vs adult Newborn vs newborn + PB 5.9 (6.3-2.6) 1.6 (2.2-1.1) 1.8 (3.4-1.2)
1.8 (2.8-1.2) 2.1 (2.7-1.6) 4.0 (7.9-2.4)
Adult vs newborn + PB Newborn + PB vs newborn 1.8 (2.4-l .4)
2.6 (3.2-2.2)
n Newborn rats 2-5 days postpartum and adult rats 120-150 g were used. Groups of lo-20 animals were used in each experimental group. Pesticides were administered ip in corn oil; phenobarbital (PB) was administered ip.
Metabolism of chlordane is considered to be a detoxification mechanism by direct replacement of chlorine by a hydroxyl group to form chlorohydrin and a diol (Wilkinson, 1968). The newborn is less sensitive to the toxic effects of chlordane than the adult (Table 1). Phenobarbital pretreatment increases the sensitivity of the newborn to chlordane toxicity. Epoxidation of unsaturated cyclodienes such as heptachlor has been shown to result from the activity of a typical microsomal enzyme requiring NADPH and oxygen (Brooks, 1965). Epoxidation is regarded as an activating step rather than detoxication. Comparative lethality data for heptachlor between adult, newborn, and phenobarbital pretreated newborn are shown in Table 1. The newborn is less sensitive to the toxic effects of heptachlor. Phenobarbital pretreatment increases the sensitivity of the newborn to heptachlor toxicity. Thus, the ability to metabolize and bioactivate heptachlor correlates with its toxicity in the newborn. The next pesticide to be considered is parathion. The microsomal enzymes cannot only oxidize parathion to the active cholinesterase inhibitor, paraoxon, but can also
SHORT
COMMUNICATION
445
detoxify parathion to p-nitrophenol and diethyl phosphorothionate. Also, hydrolysis of the phosphorous of paraoxon can yield diethyl phosphate and p-nitrophenol. Since the newborn has low microsomal mixed function oxidase activity, parathion should be less toxic in the newborn. Comparative lethality data for parathion between adult, newborn, and phenobarbital-pretreated newborn are shown in Table 1. Paradoxically, the newborn is more sensitive to the toxic effects of parathion. Phenobarbital pretreatment decreases the sensitivity of the newborn to parathion toxicity. Thus, inducing the metabolism of parathion in the newborn appears to enhance detoxification rather than bioactivation. Often, pesticides are more toxic in the newborn than the adult (Lu et al., 1965). However, of the four pesticides studied, three of these, DDT, chlordane, and heptachlor were less toxic in the newborn. DuBois and Puchala (1961), Murphy and DuBois (1958), and Brodeur and DuBois (1963; 1967) reported that most organophosphate insecticides are more toxic to weanling rats than adults. Similarly, the results presented here demonstrate parathion is more toxic in the newborn. Toxicity differences may result from differences in cholinesterase activities between the two age groups. Protection against the toxicity of various insecticides of the organophosphorous series has been reported following pretreatment with drugs known to induce hepatic microsomal enzyme activity (Ball et al., 1954; Welch and Coon, 1964; DuBois and Kinoshita, 1966). More recently, Alary and Brodeur (1969) reported phenobarbital protected against parathion toxicity in adult female rats. The data presented here demonstrates phenobarbital pretreatment potentiates the toxicity of DDT, chlordane, and heptachlor in the newborn. Phenobarbital may stimulate production of a metabolite inherently more toxic than the parent compound in newborn rats. Phenobarbital pretreatment, however, did protect against parathion toxicity. Alary and Brodeur (1969) reported phenobarbital stimulates the direct degradation of parathion to diethyl phosphorothioic acid and p-nitrophenol. Hydrolysis of paraoxon by the newborn is probably deficient. REFERENCES ALARY, J. G. AND BRODEUR, J. (1969). Studies on the mechanism of phenobarbital-induced protection against parathion in adult female rats. J. Pharmacol. Exp. Ther. 169, 159-167. BALL, W. J., SINCLAIR, J. W., CREVIEW, M. AND KAY, K. (1954). Modification of parathion’s toxicity for rats by pretreatment with chlorinated hydrocarbon insecticides. Can. J. Biochem. Physiol. 32,4%445. BRODEUR, J. AND DUBOIS,K. P. (1963). Comparison of acute toxicity of cholinesterase insecticides to weanling and adult male rats. Proc. Sot. Exp. Biol. Med. 114,509-511. BRODEUR, J. AND DUBOIS, K. P. (1967). Studies on factors influencing the acute toxicity of malathion and malaoxon in rats. Can. J. Physiol. Pharmacol. 45, 621-63 1. BROOKS, G. T. (1965). Structure activity relationship among insecticidal compounds derived from chlordane. Nature (London) 205, 1031-1032. DALE, W. E., GAINES, T. B., HAYES, W. J. AND PEARCE, G. W. (1963). Poisoning by DDT. Relation between clinical signs and concentration in rat brain. Science 142, 1474-1476. DuBors, K. P. AND PUCHALA, E. (1961). Studies on the sex difference in toxicity of a cholinergic phosphorothioate. Proc. Sot. Exp. Biol. Med. 107, 908-911. DuBors, K. P. AND KINOSHITA, F. (1966). Stimulation of detoxification of O-ethyl 0-i4nitrophenyl-phenyl-phosphonothiate (EPN) by nikethamide and phenobarbital. Proc. Sot.
Exp. Biol. Med. 121, 59-62.
446
SHORT COMMUNICATION
FINNEY, D. J. (1947). Probit Analysis. CambridgeUniversity Press. HAYES,W. J. ANDDALE,W. E. (1964). Concentration of DDT in brain and other tissuesin relation to symptomatology. Toxicol. AppE. Pharmacol. 6, 349. HENDERSON, G. L. ANDWOOLLEY,D. A. (1970). Ontogenesisof drug-inducedtremor in the rat. J. Pharmacol. Exp. Thu. 175, 113-120. Lu, F. C., JESSUP, D. C. ANDLAVALLEE,A. (1965).Toxicity of pesticidesin young versusadult rats. Fd. Cosmet. Toxicol. 3, 591-596. MURPHY,S. D. ANDDUBOIS,K. P. (1958).The influenceof various factors on the enzymatic conversion of organic thiophosphatesto anticholinesteraseagents.J. Pharmacol. Exp. Ther. 124, 194202. WELCH,R. M. ANDCOON,J. M. (1964).Studieson the effect of chlorcyclizine and other drugs on the toxicity of several organophosphateanticholinesterases. Pharmacol. Exp. Ther. 143,192-198.
WILKINSON,C. F. (1968). Detoxification of pesticidesand the mechanismof synergism.In The Enzymatic Oxidation of Toxicants (Ernest Hodgson, ed.). North Carolina State University Press,Raleigh,North Carolina. RAYMONDD. HARBISON Center in Toxicology Vanderbilt Medical Center Nashville, Tennessee 37232 Received September 20,1974;
accepted November 25,1974
