- Email: [email protected]
The effects of phenobarbital pretreatment on the metabolism and acute toxicity of the pesticide parathion in the mouse
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
86,105- 1 1 1 ( 1986)
The Effects of Phenobarbital Pretreatment on the Metabolism Toxicity of the Pesticide Parathion in the Mouse’
and Acute
LESTER G. SULTATOS Depatiment of Pharmacology, Louisiana State University Medical Center, 1901 Perdido Street, New Orleans, Louisiana 70112-I 393
Received March 31,1986; accepted June 30, I986 The Effects of Phenobarbital Pretreatment on the Metabolism and Acute Toxicity of the Pesticide Parathion in the Mouse. SULTATOS, L. G. (1986). Toxicol. Appl. Pharmacol. 86, 1051 11. Single-pass perfusion of mouse livers in situ with the phosphorothioate pesticide parathion resulted in formation of the cholinesterase inhibitor paraoxon (PO), pnitrophenol (PNP), pnitrophenyl sulfate. (PNPS), and pnitrophenyl glucuronide (PNPG). Daily pretreatment of mice with phenobarbital (80 mg/kg, ip) for 4 days induced hepatic cytochrome P-450 content, as well as oxidative activation and oxidative detoxification of parathion, as measured in vitro. However, phenobarbital pretreatment did not alter production of PO from parathion in mouse livers perfused in situ, although it increased production of PNP, PNPS, and PNPG. Additionally, phenobarbital pretreatment antagonized the acute toxicity of parathion in mice. These results indicate that phenobarbital pretreatment clearly induces that form(s) of cytochrome P450 catalyzing conversion of parathion to PO. Yet increased amounts of PO do not exit perfused livers from phenobarbital pretreated mice. Instead, the enhanced detoxification of parathion to PNP, PNPS, and PNPG likely results in the observed antagonism of parathion’s acute toxicity.
0 1986 Academic
Press, Inc.
The acute mammalian toxicity of the phosphorothioate pesticides results from their metabolism to their corresponding oxygen analogs (oxons) which are potent inhibitors of cholinesterases (Myers et al., 1952; Gage, 1953; Metcalf and March, 1953a,b). Production of these oxygen analogs occurs through cytochrome P-450-dependent oxidative desulfuration of the parent pesticides (Kulkarni and Hodgson, 1980). Pretreatment of animals with phenobarbital has been shown to increase the hepatic metabolic activation of certain phosphorothioates, as measured by in vitro techniques (Neal, 1972; Norman and ’ This research was supported by Research Grant ES 03435 from the National Institute of Environmental Health Sciences, and by a grant from the Edward G. Schlider Foundation.
Neal, 1976; Sultatos et al., 1984b). Yet the effects of phenobarbital, as well as of other enzyme inducers such as DDT, on the hepatic biotransformation and resultant toxicity of phosphorothioate pesticides in vivo are unclear. For example, phenobarbital pretreatment has been reported to both increase and decrease the acute mammalian toxicity of certain phosphorothioates (Menzer and Best, 1968; Alary and Brodeur, 1969; Sultatos et al., 1984b). Interpretation of these observations is further complicated by evidence suggesting that extrahepatic activation of certain phosphorothioate pesticides could play a dominant role in mediating their acute toxicities (Poore and Neal, 1972; Sultatos et al., 1985). A previous study from this laboratory has demonstrated that the liver likely plays a 105
0041008X/86
$3.00
Cop*&i 0 1986 by Academic Ress, Inc. AU rights of reproduction in any form reserved.
LESTER G. SULTATOS
106
dominant role in mediating the acute toxicity of the pesticide parathion in the mouse (Sultatos et al., 1985). Mouse livers perfused in situ with parathion produced the cholinesterase inhibitor paraoxon, which in turn exited the liver in significant amounts. Consequently, alterations in hepatic biotransformation of parathion brought about by pretreatment with inducers should lead to a change in parathion’s acute toxicity. The present study is directed toward investigating both the effects of phenobarbital pretreatment on the biotransformation of parathion by intact mouse livers, as well as the subsequent effects of this microsomal enzyme inducer on parathion’s acute toxicity. METHODS Chemicals. Parathion (O,Odiethyl-O-(4-nitrophenyl) phosphorothioate) and chlorpyrifos (0,O - diethyl - O(3,5,6-trichloro-2-pyridyl) phosphorothioate) were purchased from Chem Service, Inc. (West Chester, Pa.). Both were at least 98% pure as determined by HPLC (Sultatos and Minor, 1986). Paraoxon (O,Odiethyl-0-(4nitrophenyl) phosphate) (PO) was synthesized and purified by vacuum distillation as previously described (Sultatos et al., 1984a). Para-nitrophenyl sulfate (PNPS), p nitrophenol (PNP), p-nitrophenyl-D+-glucuronide (PNPG), and 4-nitrocatechol were purchased from Sigma Chemical Company (St. Louis, MO.). Glucosedphosphate, NADP, and phenobarbital were purchased from Sigma, while glucose-6-phosphate dehydrogenase and bovine serum albumin (BSA) were purchased from Boehringer - Mannheim Corporation (Indianapolis, bid.). HPLC solvents were glass-distilled or HPLC grade, while all other chemicals were reagent grade. Animals and pretreatments. Male Hla:(SW)BR Swiss Webster mice (20-30 g) obtained from Hilltop Lab Animals, Inc. (Scottdale, Pa.) were used in ah experiments. They were housed under standard laboratory conditions and had free accessto water and feed (Purina Chow). Animals were pretreated with either 0.9% saline (0.5 ml/kg, ip) or sodium phenobarbital (80 mg/kg, ip, in saline) daily for 4 days prior to the administration of a challenge dose of parathion (17 mg&, ip, in 10% DMSO in corn oil). Mice were observed for 7 days following administration of parathion. Isolation of microsomes and biochemical analyses. Mouse hepatic microsomes were isolated and measurements of microsomal activation and detoxification of
parathion were performed as described by Sultatos and Murphy (1983). Kinetic parameters were determined by direct linear plots (Eisenthal and Comish-Bowden, 1974). Cytochrome P-450 content was determined in an SLM-Aminco 2C UV-VIS recording spectrophotometer by the method of Omura and Sato ( 1964). The concentration of microsomal protein of the suspension in the cuvette was 1 mg protein/ml. Cholinesterase activities were assayed by the calorimetric methods of Ellman et al. ( I96 1), as modified by Benke et al. ( 1974). Liver perfusions. Single-pass (nonrecirculating) liver perfusions were performed as previously described (Sultatos and Minor, 1986). Mice not previously exposed to parathion were used for perfusions. Parathion was dissolved in methanol and added to the perfusate reservoir (200 ml) in volumes not greater than 100 ~1. Perfusate contained 4% BSA in all experiments. Chemical analyses. Parathion and metabolites were determined by HPLC as previously described (Diamond and Quebbemann, 1979; Sultatos and Minor, 1986). A Waters (Milford, Mass.) high-performance liquid chromatograph system was used and included two 501 pumps, a U6-K injector, an automated gradient controller, and a 481 UV-VIS detector. Quantitative analyses were made using an LDC/Milton Roy CI- 10 integrator (LDC Milton Roy, Riviera Beach, Ra.). Data analysis. Assuming that the liver acts as a wellstirred system with the effluent concentration in equilibrium with that in the liver, the equation describing the output of a single-pass perfused liver receiving a constant input of substrate can be expressed as follows: I-exp-&t co,,= CO”,,,
vh&
(1)
where C,,, is the effluent concentration of substrate at time t, CkW is the effluent substrate concentration at steady-state, Q is the rate of perfitsate flow, VH is the volume of the liver, and KP is the distribution ratio @chary and Rowland, 1983). If we define Kas
(2) then the half-life of approach of the effluent substrate to steady-state (tl12ss) is 0.693 . K
t,,+s = -
In the present study, the extraction ratios of parathion under steady-state conditions (&) were calculated from the equation
where Ci,+ represents the incoming parathion concen-
PARATHION
METABOLISM
IN PERFUSED
TABLE
107
LIVERS
1
EFFECTSOF PHENOBARBITAL PRETREATMENT ON MOUSE HEPATIC MICROSOMAL CYT~CHROME P-450 CONTENT, AND ACTIVATION AND DETOXFICATION OF PARATHION Pretreatmenta Saline Phenobarbital
Products Paraoxon pNitropheno1 Paraoxon pNitropheno1
am kmb bm)
am Vmaxb (nmol/ 100 mg liver/min)
Cytochrome P-450’ (nmol/mg microsomal protein)
24.8 t 3.6
5.3 + 0.7
0.61 f 0.14
22.1 + 5.2
6.1 + 0.9 12.3 + 1.0’ 12.9 + 1.3’
1.38 kO.21’
20.9 -t 4.1
21.8 & 3.3
0 Mice were pretreated with daily ip injections of phenobarbital (80 mg/kg) in saline, or saline alone, for 4 days prior to determinations. b Each value represents the mean * SD of four mice. c Significantly different (p < 0.05) from the saline control group by analysis of variance followed by the NewmanKeuls test.
tration and C,,,, represents the effluent parathion concentration at steady-state (Pang and Rowland, 1977). The effluent steady-state concentrations (C,,,=) were calculated by performing nonlinear regression analyses on effluent concentration versus perfusion time profiles using Eq. (I) as the basic model (PCNONLIN, Statistical Consultants, Inc., Lexington, KY.). The half-lives for ap preach to steady-state (t,,ass)were determined as secondary parameters of PCNONLIN utilizing Eq. 3. Statistical analyses were performed by a t test, the ap propriate analysis of variance, followed by the NewmanKewls test (Winer, 1971), or by X2 analysis (Goldstein, 1964).
steady-state is achieved, the difference in concentration between substrate entering the perfused liver and substrate in the effluent is due solely to biotransformation (Pang and Rowland, 1977). Following perfusion of parathion into livers from saline or phenobarbital-pretreated mice, steady-state conditions were achieved (Fig. 1). The per&sate concentrations used in these experiments were 5, 30, and 50 PM, since hepatic portal concentrations of parathion would be expected to fall within this range following ip
RESULTS Daily pretreatment of mice with phenobarbital for 4 days induced hepatic cytochrome P-450 content, as well as both hepatic oxidative activation and oxidative detoxification of parathion, as measured by in vitro techniques (Table 1). Although phenobarbital pretreatment induced activation and detoxification similarly (as measured in vitro; Table I), it antagonized the acute toxicity of parathion in mice (Table 2) without directly affecting tissue cholinesterase activities (data not shown). With single-pass liver perfusions, steadystate (or quasi steady-state) conditions with respect to a substrate are indicated by the constancy of substrate appearing in the effluent (Schary and Rowland, 1983). Once
TABLE
2
THE EFFECTS OF PHENOBARBITAL ON THE LETHALITY OF A CHALLENGE IN MICE
Pretreatments’ Experiment 1 Saline Phenobarbital Experiment 2 Saline Phenobarbital
DOSE OF PARATHION
(17 mg/kg)
Lethality 50% (4/8) 0% (0/8)b 38% (3/8) 0% (O/8)b
LIMice were pretreated with daily ip injections of phenobarbital (80 m&kg) in saline, or saline alone, for 4 days prior to the challenge with parathion. “Significantly different (p < 0.05) from the corresponding control by X2 analysis.
108
LESTER G. SULTATOS 30 1
Y 0
5
25
15 Perfusion
3'5
I
45
Timelminl
FIG. 1. Profile with time of unchanged parathion in effluent following perfusion of liver from phenobarbital-pretreated mice (0) or saline-pretreated mice (m). Parathion reservoir concentrations were 30 pM.
injections of near-lethal or lethal doses (Sultatos et al., 1985). Livers from phenobarbitalpretreated mice displayed the same half-lives (tI& for approach to steady-state as those from saline-pretreated mice (Table 3). HowTABLE 3 EFFECTS OF PHENOBARBITAL THE DISKBSITION OF PARATHION PERFUSED inSitu”
PRETREATMENT ON IN MOUSE LIVERS
Pretreatment.@ Saline Phenobarbital
5.9 + 0.7
0.27 f 0.04
6.3 k 0.9
0.50 + 0.07d
(1Reservoir concentrations of parathion were 30 PM. Additional experiments were performed using 5 and 50 MM parathion, with identical results. b Mice were pretreated daily with ip injections of phenobarbital (80 mg/kg) in saline, or saline alone, for 4 days prior to perfusions. c Each value represents the mean + SD of eight mice. d Siinificantiy different (p < 0.05) from the saline control group by the t test.
ever, livers of phenobarbital-pretreated mice had greater extraction ratios for parathion than did livers of saline controls (Table 3, Fig. 1). Phenobarbital pretreatment did not significantly increase liver wet weight (data not shown). Perfusion of mouse livers from saline and phenobarbital-pretreated mice resulted in formation of PO, PNP, PNPS, and PNPG (Table 4). Metabolism of parathion could be totally accounted for in both groups of mice through production of these metabolites (Table 4). Although phenobarbital pretreatment enhanced production of PNP, PNPS, and PNPG from livers perfused with parathion, surprisingly it had no effect on production of paraoxon (Table 4). DISCUSSION The effects of induction of one or more various forms of hepatic cytochrome P-450 on
PARATHION
METABOLISM
IN
TABLE Emcrs
PERFUSED
109
LIVERS
4
OF PHENOBARBITALPRETREATMENTON THERECWERYOF PARATHIONANDMETABOLITESIN EFFLUENT PERIWSATE AT STEADY-STATE CONLXTIONS~ Effluent per&sate’
Pretreatment@ Saline Phenobarbital
Parathiond
Paraoxond
77.8 f. 11.7 54.3 + 9.8’
3.3 r 0.3 3.4 + 0.2
pNitrophenold 2.9 + 0.4 14.0 + 0.9’
pNitrophenyld glucuronide 2.7 f 0.4 6.2 + 0.8
pNitrophenyld sulfate 10.5 + 1.1 20.3 + 2.8d
Recoveryd 6) 97.2 2 5.4 98.2 + 4.8
a Reservoir concentrations of parathion were 30 pM. Additional experiments were performed using 5 and 50 PM parathion, with identical results. * Mice were pretreated daily with ip injections of phenobarbital (80 m&kg) in saline, or saline alone, for 4 days prior to perfusions. ’ Each value represents the mean l?rSD of 10 mice. d Values are expressed as the percentage of the concentration of parathion entering the liver (30 PM). e Significantly different (p < 0.05) from the corresponding saline control group by analysis of variance followed by the Newman-Keuls test.
metabolism in vitro of phosphorothioates like parathion have been well - documented (Kulkami and Hodgson, 1980). In the present study, phenobarbital pretreatment stimulated equally both oxidative activation and oxidative detoxification of parathion by liver, as measured in vitro, confirming previous reports (Neal, 1972; Norman and Neal, 1976) although contradicting one study (Alar-y and Brodeur, 1969). Yet the difficulties in relating the effects of phenobarbital induction on parathion’s metabolism in vitro with the effects of the induction on the acute toxicity of this pesticide in vivo are evident, since phenobarbital pretreatment of mice antagonized the acute toxicity of parathion (Table 2). As pointed out by Chapman and Leibman (197 l), studies have demonstrated that alterations in the toxicity of parathion brought about by prior exposure to an inducing agent do not parallel the changes in microsomal metabolism of parathion brought about by the same inducing agent. Proposals put forth to explain this observation include preferential induction of detoxification of parathion in vivo (Alary and Brodeur, 1969), as well as the involvement of extrahepatic activation in
mediating phosphorothioate toxicity (Poore and Neal, 1972; Sultatos et al., 1984b). Clearly, the subsequent effects of enzyme induction on the acute mammalian toxicity of pesticides like parathion are complex since activation and detoxification of these pesticides by intact liver are influenced by the relative rates of activation and detoxification, the transit times within the liver, and the anatomical localization of the enzymes catalyzing these reactions. Therefore, the present study utilized the technique of mouse liver perfusions in situ in order to relate phenobarbital-induced alterations in hepatic metabolism of parathion with the subsequent antagonism of the acute toxicity of this pesticide in mice. Perfused livers from phenobarbital-pretreated mice displayed an increased capacity to metabolize parathion, compared to salinepretreated controls, as evidenced by their larger extraction ratios (Fig. 1, Table 3). However, phenobarbital pretreatment did not alter the rate of uptake of parathion into the liver (Fig. 1, Table 3). Consequently, phenobarbital pretreatment did not alter the distribution of parathion into the liver (see Eqs. (2)
110
LESTER G. SULTATOS
and (3)). It must be pointed out that the half- tion of parathion and not oxidative activalives for approach to steady-state of parathion tion in viva However, since the metabolism in the present study (Table 3) are slightly of phosphorothioates is complex, one must greater than those reported in a previous be cautious about extrapolating the results of study from this laboratory (Sultatos and Mithe present study to other organophosphates nor, 1986) as a result of the different proce- or to other animal species. dures used to determine this parameter. Sultatos and Minor (1986) used a graphical procedure, whereas the present study utilized a REFERENCES curve-fitting procedure (see Methods section). ALARY, J. G., AND BRODEUR, J. (1969). Studies on the Interestingly, the rates of appearance of mechanism of phenobarbital-induced protection against parathion in adult female rats. J. Pharmacol. paraoxon in the effluent of livers perfused Exp. Ther. 169,159-167. with parathion were the same in phenobarbiBENKE, G. M., CHEEVER, K. L., MIRER, F. E., AND tal- and saline-pretreated mice (Table 4). The MURPHY, S. D. (1974). Comparative toxicity, anticholarger extraction ratios of livers from phenoline&erase action and metabolism of methyl parathion barbital-pretreated mice can be accounted for and parathion in sunfish and mice. Toxicol. Appl. Pharmacol. 28,97-109. by greater production of PNP, PNPS, and CHAPMAN, S. K., AND LEIBMAN, K. C. (197 1). The PNPG, compared to saline controls (Table effects of chlordance, DDT, and 3-methylcholam4). The reason for this lack of effect on apthrene upon the metabolism and toxicity of diethyl-4pearance of paraoxon is unclear. However, it nitrophenyl phosphorothionate (parathion). Toxicol. must be remembered that the hepatic bioAppl. Pharmacol. l&977-987. transformation of parathion is complex, in- DIAMOND, G., AND QUEBBEMANN, A. J. (1979). Rapid separation of pnitrophenol and its glucuronide and volving several simultaneous and sequential sulfate conjugate by reverse-phase HPLC. J. Chromareactions (Sultatos and Minor, 1986). The togr. 177,368-37 1. technique of liver perfusion quantifies the net EISENTHAL, R., AND CORMSH-BOWDEN, A. (1974). The outcome of these reactions, but yields little or direct linear plot. A new graphical procedure for estimating enzyme kinetic parameters. B&hem. J. 139, no information regarding specific intermedi715-720. ary pathways. Moreover, phenobarbital preELLMAN, G. L. (1959). Tissue sullhydryl groups. Arch. treatment has been shown to increase hepatic Biochem. Biophys. 82,70-77. microsomal A-esterase activity (Alary and GAGE, J. C. (1953). A cholinesterase inhibitor derived from O,O-diethyl-o-nitrophenyl thiophosphate in vivo. Brodeur, 1969; Murphy, 1972; Sultatos et al, Biochem. J. 54,426-430. 1984b). GOLDSTEIN, A. (1964). Biostatistics: An Introductory Although livers from phenobarbital-preText. Macmillan Co., New York. treated mice have the same capacity to pro- KULKARNI, A. P., AND HODGSON, E. (1980). Metaboduce paraoxon as do livers from saline conlism of insecticides by mixed function oxidase systems. Pharmacol. Ther. 8,379-475. trols, the former have an increased capacity MENZER, R. E., AND BEST, N. H. ( 1968). Effect of phenoto produce PNP, PNPG, and PNPS from barbital on the toxicity of several organophosphorous parathion. The increased hepatic extraction compounds. Toxicol. Appi. Pharmacol. 13,37-42. of parathion as a result of phenobarbital pre- METCALF, R. L., AND MARCH, R. B. (1953a). Reversed treatment will result in enhanced elimination phase paper chromatography of parathion and related phosphate esters, Science 117,527-528. of parathion in viva, which could easily acMETCALF, R. L., AND MARCH, R. B. (1953b). Further count for the protection against parathion studies on the mode of action of organic thionophos afforded by phenobarbital induction. These phate insecticides. Ann. Entomol. Sot. Amer. 46,63results therefore support the speculation of 74. Alary and Brodeur ( 1969) that phenobarbital MURPHY, S. D. (1972). In Proceedings ofthe Conference on Degradation of Synthetic Organic Molecules in Bi@ pretreatment stimulates only the detoxifica-
PARATHION
METABOLISM
sphere, pp. 3 13-335. National Academy of Sciences, Washington, D.C. MYERS, D. K., MENDEL, B., GERSMANN, H. R., AND KETALARR, J. A. A. (1952). Oxidation of thiophosphate insecticides in the rat. Nature (London) 170, 805-807. NEAL, R. A. (1972). A comparison ofthe in vitro metabolism of parathion in the lung and liver of the rabbit. Toxicol. Appl. Pharmacol. 23, 123- 130. NORMAN, B. J., AND NEAL, R. A. (1976). Examination of the metabolism in vitro of parathion (diethyl pnitrophenyl phosphorothionate) by rat lung and brain. Biochem. Pharmacol. 25,37-45. OMURA, T., AND SATO, R. ( 1964). The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. .I. Biol. Chem. 239,23702378. PANG, K. S., AND ROWLAND, M. (1977). Hepatic clearance of drugs. I. Theoretical considerations of a “wellstirred” model and a “parallel tube” model. Influence of hepatic blood flow, plasma and blood cell binding, and the hepatocellular enzymatic activity on hepatic drug clearance. .I. Pharmacokinet. Biopharm. 5,625653. POORE, R. E., AND NEAL, R. A. (1972). Evidence for extrahepatic metabolism of parathion. Toxicol. Appl. Pharmacol. 23,759-768.
IN PERFUSED
LIVERS
111
SCHARY, W. L., AND ROWLAND, M. (1983). Protein binding and hepatic clearance: Studies with tolbutamide, a drug of low intrinsic clearance, in the isolated perfiised rat liver preparation. J. Pharmacokinet. Biopharm. 11,225-243. SULTATOS, L. G., AND MINOR, L. D. (1986). Factors affecting the biotransformation of the pesticide parathion by the isolated perfused mouse liver. Drug. Metab. Dispos. 14,2 14-220. SULTATOS, L. G., AND MURPHY, N. (1983). Kinetic analyses of the microsomal biotransformation of the phosphorothioate insecticides chlorpytifos and parathion. Fundam. Appl. Toxicol. 3, 16-2 1. SULTATOS, L. G., BASKER, K. M., SHAO, M., ANDMURPHY, S. D. (1984a). The interaction of the phosphorothioate insecticides chlorphyrifos and parathion, and their oxans with bovine serum albumin. Mol. Pharmacol. 26,99-104. SULTATOS, L. G., MINOR, L. D., AND MURPHY, S. D. ( 1985). Metabolic activation of phosphorothioate pesticides: Role ofthe liver. .I. Pharmacol. Exp. Ther. 232, 624-628. SULTATOS, L. G., SHAO, M., AND MURPHY, S. D. ( 1984b). The role of hepatic biotransformation in mediating the acute toxicity of the phosphorothionate insecticide, chlorpyrifos. Toxicol. Appl. Pharmacol. 73, 60-68. WINER, B. J. (197 I). Statistical Principles in Experimental Design, 2nd ed. McGraw-Hill, New York.
AND
APPLIED
PHARMACOLOGY
86,105- 1 1 1 ( 1986)
The Effects of Phenobarbital Pretreatment on the Metabolism Toxicity of the Pesticide Parathion in the Mouse’
and Acute
LESTER G. SULTATOS Depatiment of Pharmacology, Louisiana State University Medical Center, 1901 Perdido Street, New Orleans, Louisiana 70112-I 393
Received March 31,1986; accepted June 30, I986 The Effects of Phenobarbital Pretreatment on the Metabolism and Acute Toxicity of the Pesticide Parathion in the Mouse. SULTATOS, L. G. (1986). Toxicol. Appl. Pharmacol. 86, 1051 11. Single-pass perfusion of mouse livers in situ with the phosphorothioate pesticide parathion resulted in formation of the cholinesterase inhibitor paraoxon (PO), pnitrophenol (PNP), pnitrophenyl sulfate. (PNPS), and pnitrophenyl glucuronide (PNPG). Daily pretreatment of mice with phenobarbital (80 mg/kg, ip) for 4 days induced hepatic cytochrome P-450 content, as well as oxidative activation and oxidative detoxification of parathion, as measured in vitro. However, phenobarbital pretreatment did not alter production of PO from parathion in mouse livers perfused in situ, although it increased production of PNP, PNPS, and PNPG. Additionally, phenobarbital pretreatment antagonized the acute toxicity of parathion in mice. These results indicate that phenobarbital pretreatment clearly induces that form(s) of cytochrome P450 catalyzing conversion of parathion to PO. Yet increased amounts of PO do not exit perfused livers from phenobarbital pretreated mice. Instead, the enhanced detoxification of parathion to PNP, PNPS, and PNPG likely results in the observed antagonism of parathion’s acute toxicity.
0 1986 Academic
Press, Inc.
The acute mammalian toxicity of the phosphorothioate pesticides results from their metabolism to their corresponding oxygen analogs (oxons) which are potent inhibitors of cholinesterases (Myers et al., 1952; Gage, 1953; Metcalf and March, 1953a,b). Production of these oxygen analogs occurs through cytochrome P-450-dependent oxidative desulfuration of the parent pesticides (Kulkarni and Hodgson, 1980). Pretreatment of animals with phenobarbital has been shown to increase the hepatic metabolic activation of certain phosphorothioates, as measured by in vitro techniques (Neal, 1972; Norman and ’ This research was supported by Research Grant ES 03435 from the National Institute of Environmental Health Sciences, and by a grant from the Edward G. Schlider Foundation.
Neal, 1976; Sultatos et al., 1984b). Yet the effects of phenobarbital, as well as of other enzyme inducers such as DDT, on the hepatic biotransformation and resultant toxicity of phosphorothioate pesticides in vivo are unclear. For example, phenobarbital pretreatment has been reported to both increase and decrease the acute mammalian toxicity of certain phosphorothioates (Menzer and Best, 1968; Alary and Brodeur, 1969; Sultatos et al., 1984b). Interpretation of these observations is further complicated by evidence suggesting that extrahepatic activation of certain phosphorothioate pesticides could play a dominant role in mediating their acute toxicities (Poore and Neal, 1972; Sultatos et al., 1985). A previous study from this laboratory has demonstrated that the liver likely plays a 105
0041008X/86
$3.00
Cop*&i 0 1986 by Academic Ress, Inc. AU rights of reproduction in any form reserved.
LESTER G. SULTATOS
106
dominant role in mediating the acute toxicity of the pesticide parathion in the mouse (Sultatos et al., 1985). Mouse livers perfused in situ with parathion produced the cholinesterase inhibitor paraoxon, which in turn exited the liver in significant amounts. Consequently, alterations in hepatic biotransformation of parathion brought about by pretreatment with inducers should lead to a change in parathion’s acute toxicity. The present study is directed toward investigating both the effects of phenobarbital pretreatment on the biotransformation of parathion by intact mouse livers, as well as the subsequent effects of this microsomal enzyme inducer on parathion’s acute toxicity. METHODS Chemicals. Parathion (O,Odiethyl-O-(4-nitrophenyl) phosphorothioate) and chlorpyrifos (0,O - diethyl - O(3,5,6-trichloro-2-pyridyl) phosphorothioate) were purchased from Chem Service, Inc. (West Chester, Pa.). Both were at least 98% pure as determined by HPLC (Sultatos and Minor, 1986). Paraoxon (O,Odiethyl-0-(4nitrophenyl) phosphate) (PO) was synthesized and purified by vacuum distillation as previously described (Sultatos et al., 1984a). Para-nitrophenyl sulfate (PNPS), p nitrophenol (PNP), p-nitrophenyl-D+-glucuronide (PNPG), and 4-nitrocatechol were purchased from Sigma Chemical Company (St. Louis, MO.). Glucosedphosphate, NADP, and phenobarbital were purchased from Sigma, while glucose-6-phosphate dehydrogenase and bovine serum albumin (BSA) were purchased from Boehringer - Mannheim Corporation (Indianapolis, bid.). HPLC solvents were glass-distilled or HPLC grade, while all other chemicals were reagent grade. Animals and pretreatments. Male Hla:(SW)BR Swiss Webster mice (20-30 g) obtained from Hilltop Lab Animals, Inc. (Scottdale, Pa.) were used in ah experiments. They were housed under standard laboratory conditions and had free accessto water and feed (Purina Chow). Animals were pretreated with either 0.9% saline (0.5 ml/kg, ip) or sodium phenobarbital (80 mg/kg, ip, in saline) daily for 4 days prior to the administration of a challenge dose of parathion (17 mg&, ip, in 10% DMSO in corn oil). Mice were observed for 7 days following administration of parathion. Isolation of microsomes and biochemical analyses. Mouse hepatic microsomes were isolated and measurements of microsomal activation and detoxification of
parathion were performed as described by Sultatos and Murphy (1983). Kinetic parameters were determined by direct linear plots (Eisenthal and Comish-Bowden, 1974). Cytochrome P-450 content was determined in an SLM-Aminco 2C UV-VIS recording spectrophotometer by the method of Omura and Sato ( 1964). The concentration of microsomal protein of the suspension in the cuvette was 1 mg protein/ml. Cholinesterase activities were assayed by the calorimetric methods of Ellman et al. ( I96 1), as modified by Benke et al. ( 1974). Liver perfusions. Single-pass (nonrecirculating) liver perfusions were performed as previously described (Sultatos and Minor, 1986). Mice not previously exposed to parathion were used for perfusions. Parathion was dissolved in methanol and added to the perfusate reservoir (200 ml) in volumes not greater than 100 ~1. Perfusate contained 4% BSA in all experiments. Chemical analyses. Parathion and metabolites were determined by HPLC as previously described (Diamond and Quebbemann, 1979; Sultatos and Minor, 1986). A Waters (Milford, Mass.) high-performance liquid chromatograph system was used and included two 501 pumps, a U6-K injector, an automated gradient controller, and a 481 UV-VIS detector. Quantitative analyses were made using an LDC/Milton Roy CI- 10 integrator (LDC Milton Roy, Riviera Beach, Ra.). Data analysis. Assuming that the liver acts as a wellstirred system with the effluent concentration in equilibrium with that in the liver, the equation describing the output of a single-pass perfused liver receiving a constant input of substrate can be expressed as follows: I-exp-&t co,,= CO”,,,
vh&
(1)
where C,,, is the effluent concentration of substrate at time t, CkW is the effluent substrate concentration at steady-state, Q is the rate of perfitsate flow, VH is the volume of the liver, and KP is the distribution ratio @chary and Rowland, 1983). If we define Kas
(2) then the half-life of approach of the effluent substrate to steady-state (tl12ss) is 0.693 . K
t,,+s = -
In the present study, the extraction ratios of parathion under steady-state conditions (&) were calculated from the equation
where Ci,+ represents the incoming parathion concen-
PARATHION
METABOLISM
IN PERFUSED
TABLE
107
LIVERS
1
EFFECTSOF PHENOBARBITAL PRETREATMENT ON MOUSE HEPATIC MICROSOMAL CYT~CHROME P-450 CONTENT, AND ACTIVATION AND DETOXFICATION OF PARATHION Pretreatmenta Saline Phenobarbital
Products Paraoxon pNitropheno1 Paraoxon pNitropheno1
am kmb bm)
am Vmaxb (nmol/ 100 mg liver/min)
Cytochrome P-450’ (nmol/mg microsomal protein)
24.8 t 3.6
5.3 + 0.7
0.61 f 0.14
22.1 + 5.2
6.1 + 0.9 12.3 + 1.0’ 12.9 + 1.3’
1.38 kO.21’
20.9 -t 4.1
21.8 & 3.3
0 Mice were pretreated with daily ip injections of phenobarbital (80 mg/kg) in saline, or saline alone, for 4 days prior to determinations. b Each value represents the mean * SD of four mice. c Significantly different (p < 0.05) from the saline control group by analysis of variance followed by the NewmanKeuls test.
tration and C,,,, represents the effluent parathion concentration at steady-state (Pang and Rowland, 1977). The effluent steady-state concentrations (C,,,=) were calculated by performing nonlinear regression analyses on effluent concentration versus perfusion time profiles using Eq. (I) as the basic model (PCNONLIN, Statistical Consultants, Inc., Lexington, KY.). The half-lives for ap preach to steady-state (t,,ass)were determined as secondary parameters of PCNONLIN utilizing Eq. 3. Statistical analyses were performed by a t test, the ap propriate analysis of variance, followed by the NewmanKewls test (Winer, 1971), or by X2 analysis (Goldstein, 1964).
steady-state is achieved, the difference in concentration between substrate entering the perfused liver and substrate in the effluent is due solely to biotransformation (Pang and Rowland, 1977). Following perfusion of parathion into livers from saline or phenobarbital-pretreated mice, steady-state conditions were achieved (Fig. 1). The per&sate concentrations used in these experiments were 5, 30, and 50 PM, since hepatic portal concentrations of parathion would be expected to fall within this range following ip
RESULTS Daily pretreatment of mice with phenobarbital for 4 days induced hepatic cytochrome P-450 content, as well as both hepatic oxidative activation and oxidative detoxification of parathion, as measured by in vitro techniques (Table 1). Although phenobarbital pretreatment induced activation and detoxification similarly (as measured in vitro; Table I), it antagonized the acute toxicity of parathion in mice (Table 2) without directly affecting tissue cholinesterase activities (data not shown). With single-pass liver perfusions, steadystate (or quasi steady-state) conditions with respect to a substrate are indicated by the constancy of substrate appearing in the effluent (Schary and Rowland, 1983). Once
TABLE
2
THE EFFECTS OF PHENOBARBITAL ON THE LETHALITY OF A CHALLENGE IN MICE
Pretreatments’ Experiment 1 Saline Phenobarbital Experiment 2 Saline Phenobarbital
DOSE OF PARATHION
(17 mg/kg)
Lethality 50% (4/8) 0% (0/8)b 38% (3/8) 0% (O/8)b
LIMice were pretreated with daily ip injections of phenobarbital (80 m&kg) in saline, or saline alone, for 4 days prior to the challenge with parathion. “Significantly different (p < 0.05) from the corresponding control by X2 analysis.
108
LESTER G. SULTATOS 30 1
Y 0
5
25
15 Perfusion
3'5
I
45
Timelminl
FIG. 1. Profile with time of unchanged parathion in effluent following perfusion of liver from phenobarbital-pretreated mice (0) or saline-pretreated mice (m). Parathion reservoir concentrations were 30 pM.
injections of near-lethal or lethal doses (Sultatos et al., 1985). Livers from phenobarbitalpretreated mice displayed the same half-lives (tI& for approach to steady-state as those from saline-pretreated mice (Table 3). HowTABLE 3 EFFECTS OF PHENOBARBITAL THE DISKBSITION OF PARATHION PERFUSED inSitu”
PRETREATMENT ON IN MOUSE LIVERS
Pretreatment.@ Saline Phenobarbital
5.9 + 0.7
0.27 f 0.04
6.3 k 0.9
0.50 + 0.07d
(1Reservoir concentrations of parathion were 30 PM. Additional experiments were performed using 5 and 50 MM parathion, with identical results. b Mice were pretreated daily with ip injections of phenobarbital (80 mg/kg) in saline, or saline alone, for 4 days prior to perfusions. c Each value represents the mean + SD of eight mice. d Siinificantiy different (p < 0.05) from the saline control group by the t test.
ever, livers of phenobarbital-pretreated mice had greater extraction ratios for parathion than did livers of saline controls (Table 3, Fig. 1). Phenobarbital pretreatment did not significantly increase liver wet weight (data not shown). Perfusion of mouse livers from saline and phenobarbital-pretreated mice resulted in formation of PO, PNP, PNPS, and PNPG (Table 4). Metabolism of parathion could be totally accounted for in both groups of mice through production of these metabolites (Table 4). Although phenobarbital pretreatment enhanced production of PNP, PNPS, and PNPG from livers perfused with parathion, surprisingly it had no effect on production of paraoxon (Table 4). DISCUSSION The effects of induction of one or more various forms of hepatic cytochrome P-450 on
PARATHION
METABOLISM
IN
TABLE Emcrs
PERFUSED
109
LIVERS
4
OF PHENOBARBITALPRETREATMENTON THERECWERYOF PARATHIONANDMETABOLITESIN EFFLUENT PERIWSATE AT STEADY-STATE CONLXTIONS~ Effluent per&sate’
Pretreatment@ Saline Phenobarbital
Parathiond
Paraoxond
77.8 f. 11.7 54.3 + 9.8’
3.3 r 0.3 3.4 + 0.2
pNitrophenold 2.9 + 0.4 14.0 + 0.9’
pNitrophenyld glucuronide 2.7 f 0.4 6.2 + 0.8
pNitrophenyld sulfate 10.5 + 1.1 20.3 + 2.8d
Recoveryd 6) 97.2 2 5.4 98.2 + 4.8
a Reservoir concentrations of parathion were 30 pM. Additional experiments were performed using 5 and 50 PM parathion, with identical results. * Mice were pretreated daily with ip injections of phenobarbital (80 m&kg) in saline, or saline alone, for 4 days prior to perfusions. ’ Each value represents the mean l?rSD of 10 mice. d Values are expressed as the percentage of the concentration of parathion entering the liver (30 PM). e Significantly different (p < 0.05) from the corresponding saline control group by analysis of variance followed by the Newman-Keuls test.
metabolism in vitro of phosphorothioates like parathion have been well - documented (Kulkami and Hodgson, 1980). In the present study, phenobarbital pretreatment stimulated equally both oxidative activation and oxidative detoxification of parathion by liver, as measured in vitro, confirming previous reports (Neal, 1972; Norman and Neal, 1976) although contradicting one study (Alar-y and Brodeur, 1969). Yet the difficulties in relating the effects of phenobarbital induction on parathion’s metabolism in vitro with the effects of the induction on the acute toxicity of this pesticide in vivo are evident, since phenobarbital pretreatment of mice antagonized the acute toxicity of parathion (Table 2). As pointed out by Chapman and Leibman (197 l), studies have demonstrated that alterations in the toxicity of parathion brought about by prior exposure to an inducing agent do not parallel the changes in microsomal metabolism of parathion brought about by the same inducing agent. Proposals put forth to explain this observation include preferential induction of detoxification of parathion in vivo (Alary and Brodeur, 1969), as well as the involvement of extrahepatic activation in
mediating phosphorothioate toxicity (Poore and Neal, 1972; Sultatos et al., 1984b). Clearly, the subsequent effects of enzyme induction on the acute mammalian toxicity of pesticides like parathion are complex since activation and detoxification of these pesticides by intact liver are influenced by the relative rates of activation and detoxification, the transit times within the liver, and the anatomical localization of the enzymes catalyzing these reactions. Therefore, the present study utilized the technique of mouse liver perfusions in situ in order to relate phenobarbital-induced alterations in hepatic metabolism of parathion with the subsequent antagonism of the acute toxicity of this pesticide in mice. Perfused livers from phenobarbital-pretreated mice displayed an increased capacity to metabolize parathion, compared to salinepretreated controls, as evidenced by their larger extraction ratios (Fig. 1, Table 3). However, phenobarbital pretreatment did not alter the rate of uptake of parathion into the liver (Fig. 1, Table 3). Consequently, phenobarbital pretreatment did not alter the distribution of parathion into the liver (see Eqs. (2)
110
LESTER G. SULTATOS
and (3)). It must be pointed out that the half- tion of parathion and not oxidative activalives for approach to steady-state of parathion tion in viva However, since the metabolism in the present study (Table 3) are slightly of phosphorothioates is complex, one must greater than those reported in a previous be cautious about extrapolating the results of study from this laboratory (Sultatos and Mithe present study to other organophosphates nor, 1986) as a result of the different proce- or to other animal species. dures used to determine this parameter. Sultatos and Minor (1986) used a graphical procedure, whereas the present study utilized a REFERENCES curve-fitting procedure (see Methods section). ALARY, J. G., AND BRODEUR, J. (1969). Studies on the Interestingly, the rates of appearance of mechanism of phenobarbital-induced protection against parathion in adult female rats. J. Pharmacol. paraoxon in the effluent of livers perfused Exp. Ther. 169,159-167. with parathion were the same in phenobarbiBENKE, G. M., CHEEVER, K. L., MIRER, F. E., AND tal- and saline-pretreated mice (Table 4). The MURPHY, S. D. (1974). Comparative toxicity, anticholarger extraction ratios of livers from phenoline&erase action and metabolism of methyl parathion barbital-pretreated mice can be accounted for and parathion in sunfish and mice. Toxicol. Appl. Pharmacol. 28,97-109. by greater production of PNP, PNPS, and CHAPMAN, S. K., AND LEIBMAN, K. C. (197 1). The PNPG, compared to saline controls (Table effects of chlordance, DDT, and 3-methylcholam4). The reason for this lack of effect on apthrene upon the metabolism and toxicity of diethyl-4pearance of paraoxon is unclear. However, it nitrophenyl phosphorothionate (parathion). Toxicol. must be remembered that the hepatic bioAppl. Pharmacol. l&977-987. transformation of parathion is complex, in- DIAMOND, G., AND QUEBBEMANN, A. J. (1979). Rapid separation of pnitrophenol and its glucuronide and volving several simultaneous and sequential sulfate conjugate by reverse-phase HPLC. J. Chromareactions (Sultatos and Minor, 1986). The togr. 177,368-37 1. technique of liver perfusion quantifies the net EISENTHAL, R., AND CORMSH-BOWDEN, A. (1974). The outcome of these reactions, but yields little or direct linear plot. A new graphical procedure for estimating enzyme kinetic parameters. B&hem. J. 139, no information regarding specific intermedi715-720. ary pathways. Moreover, phenobarbital preELLMAN, G. L. (1959). Tissue sullhydryl groups. Arch. treatment has been shown to increase hepatic Biochem. Biophys. 82,70-77. microsomal A-esterase activity (Alary and GAGE, J. C. (1953). A cholinesterase inhibitor derived from O,O-diethyl-o-nitrophenyl thiophosphate in vivo. Brodeur, 1969; Murphy, 1972; Sultatos et al, Biochem. J. 54,426-430. 1984b). GOLDSTEIN, A. (1964). Biostatistics: An Introductory Although livers from phenobarbital-preText. Macmillan Co., New York. treated mice have the same capacity to pro- KULKARNI, A. P., AND HODGSON, E. (1980). Metaboduce paraoxon as do livers from saline conlism of insecticides by mixed function oxidase systems. Pharmacol. Ther. 8,379-475. trols, the former have an increased capacity MENZER, R. E., AND BEST, N. H. ( 1968). Effect of phenoto produce PNP, PNPG, and PNPS from barbital on the toxicity of several organophosphorous parathion. The increased hepatic extraction compounds. Toxicol. Appi. Pharmacol. 13,37-42. of parathion as a result of phenobarbital pre- METCALF, R. L., AND MARCH, R. B. (1953a). Reversed treatment will result in enhanced elimination phase paper chromatography of parathion and related phosphate esters, Science 117,527-528. of parathion in viva, which could easily acMETCALF, R. L., AND MARCH, R. B. (1953b). Further count for the protection against parathion studies on the mode of action of organic thionophos afforded by phenobarbital induction. These phate insecticides. Ann. Entomol. Sot. Amer. 46,63results therefore support the speculation of 74. Alary and Brodeur ( 1969) that phenobarbital MURPHY, S. D. (1972). In Proceedings ofthe Conference on Degradation of Synthetic Organic Molecules in Bi@ pretreatment stimulates only the detoxifica-
PARATHION
METABOLISM
sphere, pp. 3 13-335. National Academy of Sciences, Washington, D.C. MYERS, D. K., MENDEL, B., GERSMANN, H. R., AND KETALARR, J. A. A. (1952). Oxidation of thiophosphate insecticides in the rat. Nature (London) 170, 805-807. NEAL, R. A. (1972). A comparison ofthe in vitro metabolism of parathion in the lung and liver of the rabbit. Toxicol. Appl. Pharmacol. 23, 123- 130. NORMAN, B. J., AND NEAL, R. A. (1976). Examination of the metabolism in vitro of parathion (diethyl pnitrophenyl phosphorothionate) by rat lung and brain. Biochem. Pharmacol. 25,37-45. OMURA, T., AND SATO, R. ( 1964). The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. .I. Biol. Chem. 239,23702378. PANG, K. S., AND ROWLAND, M. (1977). Hepatic clearance of drugs. I. Theoretical considerations of a “wellstirred” model and a “parallel tube” model. Influence of hepatic blood flow, plasma and blood cell binding, and the hepatocellular enzymatic activity on hepatic drug clearance. .I. Pharmacokinet. Biopharm. 5,625653. POORE, R. E., AND NEAL, R. A. (1972). Evidence for extrahepatic metabolism of parathion. Toxicol. Appl. Pharmacol. 23,759-768.
IN PERFUSED
LIVERS
111
SCHARY, W. L., AND ROWLAND, M. (1983). Protein binding and hepatic clearance: Studies with tolbutamide, a drug of low intrinsic clearance, in the isolated perfiised rat liver preparation. J. Pharmacokinet. Biopharm. 11,225-243. SULTATOS, L. G., AND MINOR, L. D. (1986). Factors affecting the biotransformation of the pesticide parathion by the isolated perfused mouse liver. Drug. Metab. Dispos. 14,2 14-220. SULTATOS, L. G., AND MURPHY, N. (1983). Kinetic analyses of the microsomal biotransformation of the phosphorothioate insecticides chlorpytifos and parathion. Fundam. Appl. Toxicol. 3, 16-2 1. SULTATOS, L. G., BASKER, K. M., SHAO, M., ANDMURPHY, S. D. (1984a). The interaction of the phosphorothioate insecticides chlorphyrifos and parathion, and their oxans with bovine serum albumin. Mol. Pharmacol. 26,99-104. SULTATOS, L. G., MINOR, L. D., AND MURPHY, S. D. ( 1985). Metabolic activation of phosphorothioate pesticides: Role ofthe liver. .I. Pharmacol. Exp. Ther. 232, 624-628. SULTATOS, L. G., SHAO, M., AND MURPHY, S. D. ( 1984b). The role of hepatic biotransformation in mediating the acute toxicity of the phosphorothionate insecticide, chlorpyrifos. Toxicol. Appl. Pharmacol. 73, 60-68. WINER, B. J. (197 I). Statistical Principles in Experimental Design, 2nd ed. McGraw-Hill, New York.