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Gestational Exposure to Chlorpyrifos: Comparative Distribution of Trichloropyridinol in the Fetus and Dam
Toxicology and Applied Pharmacology 158, 16 –23 (1999) Article ID taap.1999.8689, available online at http://www.idealibrary.com on
Gestational Exposure to Chlorpyrifos: Comparative Distribution of Trichloropyridinol in the Fetus and Dam D. L. Hunter,* ,1 T. L. Lassiter,* ,† and S. Padilla* *Cellular and Molecular Toxicology Branch, Neurotoxicology Division, National Health Effects and Ecological Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711; and †Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina Received January 26, 1999; accepted April 14, 1999
signs and symptoms characteristic of overstimulation of the cholinergic nervous system, presumably because of accumulation of the neurotransmitter acetylcholine in the synaptic cleft (as reviewed by Fukuto, 1990; Ecobichon, 1996). In the developing nervous system, acetylcholine, acetylcholinesterase, and butyrylcholinesterase (EC 3.1.1.8), all molecules that are likely to be affected by anticholinesterases, are postulated to play developmental roles that are unique from their ultimate function in the adult nervous system (as reviewed by Karczmar et al., 1973; Layer and Willbold, 1995; Small et al., 1996; Brimijoin and Koenigsberger, 1999). Consequently, basic research is needed to characterize the distribution of an anticholinesterase insecticide to determine if that insecticide actually reaches the fetal nervous system during brain development and to compare the concentration measured in the fetal brain to the level in the maternal brain. Chlorpyrifos (Dursban, Lorsban, O,O9-diethyl O-[3,5,6trichloro-2-pyridyl] phosphorothionate) is a widely used organophosphorus insecticide, both in agricultural and non-agricultural arenas (Cochran et al., 1995; Aspelin, 1997). Previous studies in our laboratory as well as others have shown that neonatal rats are much more sensitive to the acute effects of this insecticide than are adults (Pope et al., 1991; Moser and Padilla, 1998; Moser et al., 1998). Previous investigators believed that the fetus was not as affected by gestational exposure to anticholinesterases because the prenatal rat has the mother’s physiology to “protect” it from toxicity (Michalek et al., 1985; Santhoshkumar and Shivanandappa, 1994; Chanda et al., 1995; Chanda and Pope, 1996). In our studies of the prenatal rat, however, we have found that the fetal compartment is not protected from cholinesterase inhibition when the pregnant dam is dosed orally with chlorpyrifos. In fact, the fetal brain shows as much cholinesterase inhibition after one oral dose of chlorpyrifos to the dam as does the maternal brain (Lassiter et al., 1998). One shortcoming of our previous study was the lack of toxicokinetic data on the distribution of chlorpyrifos and its metabolites. This information is integral to gaining a better understanding of the toxicodynamic and toxicokinetic parameters describing chlorpyrifos intoxication of the pregnant rat and fetus.
Gestational Exposure to Chlorpyrifos: Comparative Distribution of Trichloropyridinol in the Fetus and Dam. Hunter, D. L., Lassiter, T. L., and Padilla, S. (1999). Toxicol. Appl. Pharmacol. 158, 16 –23. Chlorpyrifos (O,O*-diethyl O-[3,5,6-trichloro-2-pyridyl] phosphorothionate) is a commonly used anticholinesterase insecticide, and therefore the potential for human exposure is high. The present time course and dose response studies were conducted to delineate the toxicokinetics of chlorpyrifos and its metabolites in the pregnant rat and fetus. Time-pregnant, Long–Evans rats were treated orally with chlorpyrifos during late gestation (Gestational Days 14 –18). Following euthanasia the level of chlorpyrifos and its metabolites, chlorpyrifos-oxon and 3,5,6-trichloro-2-pyridinol (TCP), were measured in both fetal and maternal brain and liver (limits of quantitation: 59.2, 28.8, and 14.0 ng/g tissue, respectively). In addition, cholinesterase inhibition was also measured in the same tissues for comparison. TCP was the only component detected. The highest level of TCP and the lowest level of cholinesterase activity showed the same time of peak effect: 5 h after the last dose. The concentration of TCP in the maternal liver was approximately fivefold higher than the TCP concentration in fetal liver, but, paradoxically, the concentration of TCP in the fetal brain was two- to fourfold higher than the TCP concentration in the maternal brain. The half-life of the TCP was identical in all tissues examined (12–15 h). These toxicokinetic results suggest that the fetal nervous system may be exposed to a higher concentration of chlorpyrifos than the maternal nervous system when the dam is orally exposed to chlorpyrifos during late gestation. Key Words: fetus; chlorpyrifos; chlorpyrifos-oxon; trichloropyridinol; pregnancy.
Anticholinesterase insecticides are thought to mediate toxicity primarily through inhibition of an esterase, acetylcholinesterase (EC 3.1.1.2). The inhibition of this enzyme leads to 1 To whom correspondence should be addressed: Cellular and Molecular Toxicology Branch, Neurotoxicology Division, Mail Drop 74-B, National Health Effects and Ecological Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711. Fax: (919) 541-5024; E-mail: [email protected].
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TOXICOKINETICS OF CHLORPYRIFOS IN THE PREGNANT RAT
The present group of studies was conducted to assess and compare the distribution of chlorpyrifos and its metabolites in pregnant rats dosed with chlorpyrifos. Some of the dosing scenarios used in this group of studies replicate those employed in the previous study (i.e., Lassiter et al., 1998). The current studies were designed to monitor the concentration of chlorpyrifos and its metabolites in critical tissues in the fetus and dam. In brief, pregnant dams were orally dosed with chlorpyrifos for 5 days during late gestation, and the amounts of chlorpyrifos, chlorpyrifos-oxon (the potent anticholinesterase metabolite), and 3,5,6-trichloro-2-pyridinol (TCP, 2 the “leaving group”, see Eto, 1974) were quantified in fetal and maternal brain and liver; cholinesterase inhibition was monitored in the same tissues for comparison. Both time course and dose response assessments were conducted. The results support and extend our original observation that the fetal nervous system is not protected from chlorpyrifos toxicity and, in fact, more of this insecticide may be distributed to the fetal nervous system than to the maternal nervous system. MATERIALS AND METHODS Animals, Treatment, and Sample Collection Experimental design. Time-pregnant, primiparous Long–Evans rats were obtained from Charles River Laboratories Crl: (LE) BR (Portage, ME) and were determined to be sperm-positive on Gestational Day 0 (GD0). Dams arrived on GD4 and were housed individually on a 12:12 h light:dark cycle, with food (Rodent Diet 5001; Lab Diet, Indianapolis, IN) and water provided ad libitum. Weight gain was monitored from arrival to euthanasia. Dams were distributed among groups on GD13 according to a ranking of body weight gain to ensure balanced body weights among groups. Following chlorpyrifos exposure, weight gain also was used as a general index of maternal health status. For the time course study, groups of dams received 0 or 7 mg/kg of chlorpyrifos (dose volume: 1 ml/kg) on GD14 –18 and were euthanized at 2, 5, 10 (GD18), 24 (GD19), or 48 (GD20) h after the last dose (n/dose/time point 5 4). Additional dams and pups were euthanized at 120 h (Postnatal Day 1, P1) after the last dose. The day of birth was considered P0. Similarly, the dams in the dose response study were dosed on GD14 –18 by gavage with 0, 3, or 7 mg/kg chlorpyrifos in corn oil (n 5 4/dose group). All dose response animals, however, were euthanized at 5 h after the last dose (time of peak concentration of TCP). Sample collection. Tissue collection and analysis were the same for time course and dose response portions of this study. All dams and pups were anesthetized with CO 2 and euthanized by decapitation for collection of tissue samples. The dams were perfused transcardially with saline. The perfusion continued until the normally dark liver was a very pale brown. At each time point dam liver and brain were collected and frozen on dry ice. Half of the brain was used for chromatography measurements and half was used for assay of cholinesterase activity. Separate liver samples were collected as well. The uterus was removed, placed on ice, and the position of any resorptions recorded. Sampling roughly every other fetus in the uterus, a total of eight fetuses was removed. From each litter, two fetal livers and brains were collected individually for cholinesterase assays while the other six sets were pooled for HPLC analysis. All samples were immediately frozen on dry ice. Tissues were stored at 280°C until analysis. Litter was considered the smallest
2
Abbreviations used: ANOVA, analysis of variance; GD, gestational day; LOQ, limits of quantitation; P, postnatal day; TCP, 3,5,6-trichloro-2-pyridinol.
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unit of analysis, meaning that end points recorded for individual fetuses from a single litter were averaged as replicates. Toxicokinetic Analysis Sample preparation. All tissues for HPLC analysis were diluted using 0.1 M Na phosphate buffer (pH 7.4) and homogenized on ice (Setting 6, Polytron, Brinkman Industries, Westbury, NY). All tissues were diluted 1:3 (w/v: e.g., 1 g of tissue in 2 ml of buffer), with the exception of fetal brain, which was diluted 1:1.05. Tissue homogenates were extracted using a method based on Sultatos et al. (1982). One milliliter of tissue homogenate was added to a tube containing 1 g of NaCl; chlorpyrifos-methyl was added as an internal standard. The extractant ethyl acetate (4 ml) was added next and the samples were then vortexed for 5 min (Setting 7, multitube vortexer, Dade International, Miami, FL). The samples were then centrifuged for 10 min at 1000g (HNS-centrifuge, Damon, Needham Heights, MA). After the ethyl acetate layer was transferred to a collection tube, a second volume of ethyl acetate was added to the pellet and a second extraction was performed. Following addition of the second ethyl acetate layer to the collection tube, a gentle stream of nitrogen gas was used to evaporate the ethyl acetate. The fully dried extracts were reconstituted in 200 ml of the tissue-appropriate mobile phase. Chromatography. Analyses were made using a system manufactured by Waters (Milford, MA) consisting of a 717plus Autosampler, a 600 Solvent Delivery System, and a 996 PDA detector. The column used was a mBondapak C18 column (300 mm long 3 3.9 mm id). The mobile phase appropriate for brain samples was 71.5% methanol, 28.5% HPLC-grade water, with the pH adjusted to 3.4 using glacial acetic acid. For analysis of liver the mobile phase was 60% acetonitrile, 40% HPLC-grade water (pH 3.4). The volume of sample injected was 50 ml, the flow rate was 1.5 ml/min, and the wavelength was 290 nm. A calibration curve was established using TCP, chlorpyrifos-oxon, and chlorpyrifos standards, ranging in concentration from 5 to 500 ng, which were dissolved in the appropriate mobile phase. Spiking of control samples revealed that TCP and chlorpyrifos-oxon were recovered at 95% and recovery of chlorpyrifos was 80%. The limits of quantitation (LOQ) were set at 10 times the baseline noise. The LOQ were 14, 29, and 59 ng/g of tissue for TCP, chlorpyrifos-oxon, and chlorpyrifos, respectively. The components of interest were quantified using peak heights. A representative chromatogram of prepared standard solutions of chlorpyrifos and its metabolites is presented in Fig. 1. There was concern that some TCP may have been conjugated to glucuronide by Phase II detoxification systems. Therefore, prior to analyzing any samples, a Glusulase control experiment was performed based on a method described by Reinke et al. (1982). Homogenates of liver from chlorpyrifos-treated dams (1 ml, 1:3) were incubated with and without 10 ml of Glusulase (900 U of glucuronidase, 135 U of sulfatase, New England Nuclear, Boston, MA) for 90 min at room temperature. The samples were then extracted as described previously. The amount of TCP quantified in the Glusulase-treated and parallel control samples was the same (Fig. 2). This control experiment established that Glusulase treatment was not necessary in the preparation of the experimental samples. Biochemical Analysis Sample homogenization. Samples were homogenized (larger samples, . 50 mg wet wt: 20 s, Setting 6, Polytron) or sonicated on ice (smaller samples, , 50 mg wet wt: 10 s, Setting 3, Branson Sonifier 250) in 0.1 M Na phosphate buffer (pH 8) containing 1% Triton X-100. Maternal liver was homogenized for 45 s due to the high collagen content. Activity for the chlorpyrifos-treated subjects was expressed as percent inhibition. Cholinesterase activity. Total cholinesterase activity (acetylcholinesterase and butyrylcholinesterase) was measured for dam brain and fetal brain using an Hitachi 911 Automatic Analyzer (Boehringer Mannheim Corp., Indianapolis, IN) according to the method outlined in Hunter et al. (1997). The 911 is a spectrophotometer-equipped robot that dispenses sample, buffer, chromogen, and substrate to assay cholinesterase activity according to a variation of the
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FIG. 1. Representive chromatogram of chlorpyrifos and metabolites. The peaks are A, TCP; B, chlorpyrifos-oxon; C, chlorpyrifos-methyl (internal standard); and D, chlorpyrifos.
Ellman et al. (1961) method. Considering the high sulfhydryl background present in the liver and resultant reaction with the chromogen used in the Ellman assay, it was prohibitive to use this colorimetric technique. Fetal and maternal liver cholinesterase activity, therefore, was assayed using the Johnson and Russell (1975) radiometric assay with a final substrate concentration of 1.2 mM. The [ 3H]acetate produced in the hydrolysis of [ 3H]acetylcholine was quantified using a Wallac 1410 liquid scintillation counter.
Statistics In the time course study, the cholinesterase inhibition and TCP concentration were analyzed the same way. A two-way analysis of variance (ANOVA) (treatment vs time) was performed on each of the four tissues (fetal and maternal brain and liver). Step-down, one-way ANOVAs were then used to determine the treatment-related effects across the time course (SAS Institute, 1989). In the dose response study, the statistical analysis of cholinesterase inhibition data and the TCP concentration data was the same. For each of the four tissues (fetal and maternal brain and liver), a one-way ANOVA was used to determine if there were statistical differences among any of the treatment groups. In all cases, the dose effect was significant. Tukey’s multiple comparison procedure was used to determine which dose groups were different. The T21 of TCP was calculated based on a straight line fitted to the log-transformed TCP data for the 5, 10, 24, and 48 h time points.
RESULTS
Glusulase Treatment
FIG. 2. Glusulase treatment. Parallel sets of samples were prepared with or without Glusulase treatment. Glusulase treatment was intended to liberate TCP that had been conjugated to glucuronide. The concentration of TCP in maternal liver was unaffected by pretreatment with Glusulase.
To determine if glucuronide conjugates of TCP had been formed, liver tissue from chlorpyrifos-treated dams was incubated with Glusulase, which would hydrolyze the conjugates and produce free TCP, which could be measured. A corresponding aliquot of the same liver homogenate that was not treated with Glusulase was extracted concurrently. The levels of TCP were measured in both samples and the results compared in Fig. 2. There was no difference in TCP concentrations
TOXICOKINETICS OF CHLORPYRIFOS IN THE PREGNANT RAT
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whether the sample was treated with Glusulase, which indicated that glucuronide-conjugates of TCP were not present in adult rat liver. Maternal liver was chosen because, among the tissues sampled, it is the one most likely to glucuronidate TCP (Lucier, 1976; Dutton, 1978; Juchau et al., 1980; Dutton and Leakey, 1981; Dvorchik et al., 1986; Suleman et al., 1993, 1998; Martinasevic et al., 1998). Because the Glusulase treatment had no effect on maternal liver TCP concentrations, Glusulase treatment of the other samples was not necessary. Overt Toxicity No overt maternal toxicity was noted in either the time course or dose response studies. The rate of weight gain for controls and chlorpyrifos-treated dams was comparable (data not shown). Furthermore, gestational chlorpyrifos treatment did not affect fetal body/brain weights, number of resorptions, or litter size (data not shown). Toxicokinetic Analysis In the time course study, pregnant rats were dosed with chlorpyrifos (0 or 7 mg/kg/day) on GD14 –18 and euthanized at various intervals after the last dose. Livers collected from dams and fetuses were analyzed for the presence of chlorpyrifos and its metabolites using HPLC. The only analyte present in measurable quantities was TCP. The TCP concentration time course indicated that, while maximal concentrations of TCP were measured 5 h after the last dose for both maternal and fetal liver, the maternal liver contained fivefold more TCP than the fetal liver at this time point (Fig. 3A). TCP levels decreased sharply after the 5-h time point in both maternal and fetal liver and were below the LOQ 48 h postdosing in the fetus and by P1 in the dams. The analysis of brain tissue from the time course study produced some unexpected results. The fetal brain TCP concentration was twice as high as the maternal brain concentration 5 h after the last dose of chlorpyrifos (the peak of TCP concentration; Fig. 3B). TCP levels decreased sharply after the 5-h time point in both the dam and fetal brain, with no measurable TCP in either tissue 48 h after the last dose of chlorpyrifos. In the dose response study, pregnant rats were treated with chlorpyrifos (0, 3, or 7 mg/kg/day) on GD14 –18 and euthanized 5 h after the last dose. As in the time course study, maternal and fetal brain and liver were analyzed for chlorpyrifos and its metabolites. Again, TCP was the only metabolite present in measurable quantities, with maternal liver concentrations much higher than fetal liver levels for both the 3 and 7 mg/kg/day treatments: 8.0- and 6.6-fold, respectively (Fig. 4A). For both the maternal and fetal liver, the 7 mg/kg/day treatment resulted in higher TCP levels than the 3 mg/kg/day treatment (Fig. 4A). For the brain, the unusual results revealed in the time course study were replicated in the dose response study. TCP levels
FIG. 3. Time course of trichloropyridinol in the liver and brain. (A) TCP levels in the liver were highest 2–10 h after the last 7 mg/kg gestational dose of chlorpyrifos. The amount of TCP in the maternal liver was elevated above the fetal liver amount for at least 48 h after the last dose of chlorpyrifos. }, Statistically significant increase in TCP between the fetal and maternal tissue, at that particular time point. The areas under the curve were 6164 and 32,147 ng 3 day/g of tissue for fetal and maternal liver, respectively. The T21 of TCP in the fetal and maternal liver was comparable after repeated, gestational exposure: 15.4 and 14.0 h, respectively. The LOQ is indicated by the dotted horizontal line. (B) Similar to TCP levels in the liver, TCP in the fetal and maternal brain were highest 2–10 h after the last gestational dose of chlorpyrifos. Most importantly, at 2 and 5 h after the last dose there was more than twice as much TCP in the fetal brain as in the maternal brain. The areas under the curve were 2783 and 1,768 ng 3 day/g of tissue for fetal and maternal brain, respectively. T21 of TCP was 12.5 and 14.8 h for fetal and maternal brain, respectively.
were much higher in the fetal brain than in the maternal brain (225 vs 76 ng/g in the 7 mg/kg/day group and 123 vs 36 ng/g in the 3 mg/kg/day group; Fig. 4B). In the fetal brain, the 7 mg/kg/day treatment caused a higher concentration of TCP than the 3 mg/kg dosage level. The amount of TCP in the maternal brain was comparable at both dosage levels (Fig. 4B). Cholinesterase Inhibition Cholinesterase activity levels were determined in liver and brain from maternal and fetal rats in both the time course and
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dose of chlorpyrifos, but the maternal brain cholinesterase activity was still inhibited 44% on P1, 5 days after the last dose. The results of the time course study were replicated in the dose response, as there was more cholinesterase inhibition in maternal liver than in fetal liver (3.5- and 1.5-fold, 3 and 7 mg/kg/day treatments, respectively; Fig. 5C). Cholinesterase inhibition was similar for both chlorpyrifos treatments in the maternal liver (84 vs 89%, 3 vs 7 mg/kg/day, respectively), but the fetal liver cholinesterase activity was more inhibited by the 7 mg/kg/day treatment than the lower treatment level (Fig. 5C). The dose response results for brain tissue indicated that there was also much more cholinesterase inhibition in the maternal brain than in the fetal brain: 87 vs 32% in the 7 mg/kg/day group and 41 vs 3% in the 3 mg/kg/day group (Fig. 5D). In both maternal and fetal brain, the 7 mg/kg/day treatment resulted in more cholinesterase inhibition than the 3 mg/kg/day dosage of chlorpyrifos (Fig. 5D). DISCUSSION
FIG. 4. Dose response of trichloropyridinol in the liver and brain. (A) Similar to the time course, there was more TCP in the maternal liver than fetal liver at both dosage levels. The 7 mg/kg dosage of chlorpyrifos caused higher levels of TCP in the maternal and fetal liver than the 3 mg/kg dosage. *Statistically significant increase in TCP at the higher dose; in the same tissue, }, statistically significant difference in TCP concentration between the fetal and maternal tissue, at that particular dose. The LOQ is indicated by the dotted horizontal line. (B) Following repeated, gestational chlorpyrifos exposure, there was more TCP in the fetal brain than the maternal brain: 4.5- and 3.0-fold more at 3 and 7 mg/kg chlorpyrifos, respectively. Repeated exposure to 7 mg/kg chlorpyrifos also resulted in more TCP in the fetal brain than exposure to the 3 mg/kg dosage level. The amount of TCP in the maternal brain was comparable at both dosages. When the levels are compared across tissue, the maternal liver has more TCP than the maternal brain at both 3 and 7 mg/kg, but this is not true for the fetal tissues: the amount of TCP in the fetal brain is comparable to the amount in the fetal liver at 3 or 7 mg/kg.
dose response studies. In the time course study the time span of maximal cholinesterase inhibition in all tissues was 5–10 h after the last gestational dose of chlorpyrifos. Cholinesterase inhibition recovered after the 5–10-h time points with fetal levels returning to normal sooner than dam levels. Maternal liver cholinesterase was twofold more inhibited than fetal liver activity (Fig. 5A). Similarly, the maternal brain exhibited more cholinesterase inhibition than fetal brain at 5 h after the last dose of chlorpyrifos (84 vs 25%; Fig. 5B). Fetal brain cholinesterase recovered to control levels by 24 h after the last
The results of the present group of studies imply that the fetus is not protected from chlorpyrifos when the dam is dosed orally; these toxicokinetic results parallel the toxicodynamic results reported in a previous group of experiments (Lassiter et al., 1998). In fact, in the present study, the fetal brain received a higher dose of TCP than did the maternal brain. These higher levels of TCP were not a result of increased accumulation within the fetal compartment, i.e., that the TCP was created in and was unable to leave the fetal compartment. If that were the case, the T12 of TCP in the fetal tissues would be much longer than in the maternal tissues, but the T21s for TCP in maternal and fetal tissues were not different. The calculated T21 for maternal or fetal brain or liver was 12–15 h, which agrees well with previously published data for rat liver (Smith et al., 1967). There are three logical explanations as to why only TCP was detected in the liver and brain: (1) TCP had the lowest limit of quantitation; (2) chlorpyrifos-oxon was not detected because it is an extremely ephemeral molecule that is rarely detected in tissues of animals treated in vivo with chlorpyrifos (e.g., Ivey et al., 1972; Mann et al., 1973; McKellar et al., 1976; Barron et al., 1991; Drevenkar et al., 1993), except fat (Claborn et al., 1968), or even after in vitro “spiking” of tissues (Ivey et al., 1972; Dishburger et al., 1977; Brzak et al., 1998); and (3) chlorpyrifos is rarely detected in any tissue except blood (Drevenkar et al., 1993; Nolan et al., 1984) or fatty tissue (Claborn et al., 1968; McKellar et al., 1976; Ivey et al., 1978; Ivey and Palmer, 1979) and usually after a relatively high dose exposure. The TCP could have arisen from either a detoxification (e.g., hydrolysis by P450s or A-esterases, or binding to carboxylesterases; Sultatos et al., 1985) or toxic reaction (e.g., binding of chlorpyrifos-oxon to cholinesterase). Because of the low levels of detoxification enzymes in the brain tissue, TCP in the brain probably arose from the binding of the oxon to cholinesterase or through TCP in the circulation. More than
TOXICOKINETICS OF CHLORPYRIFOS IN THE PREGNANT RAT
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FIG. 5. Time course and dose response of cholinesterase inhibition in the liver and brain. (A) Fetal liver cholinesterase was maximally inhibited 2–5 h after the last dose of chlorpyrifos (43% inhibition), while maternal liver cholinesterase was more than 80% inhibited at 2, 5, and 10 h after the last treatment. There was recovery of fetal liver cholinesterase activity by 10 h after the last dose, but cholinesterase in the maternal liver did not recover until 48 h after the last chlorpyrifos exposure. }, Statistically significant increase in cholinesterase inhibition between the fetal and maternal tissue, at that particular dose. (B) Maximal cholinesterase inhibition occurred at 5–10 h after the last dose of chlorpyrifos. There was, however, 4.7-fold more inhibition in the maternal brain than in the fetal brain. Fetal brain cholinesterase inhibition recovered by 24 h after the last dose, but maternal brain activity did not recover completely within the days sampled (GD18 –P1). (C) Fetal liver cholinesterase activity demonstrated dose-dependent inhibition at 3 and 7 mg/kg. *Statistically significant increase in cholinesterase inhibition at the higher dose, in the same tissue. Maternal liver cholinesterase activity was maximally inhibited at both dosage levels. (D) Fetal brain cholinesterase activity was not inhibited at the lower dose, but gestational exposure to the 7 mg/kg dosage did cause inhibition. Maternal brain cholinesterase was inhibited in a dose dependent fashion at 3 and 7 mg/kg chlorpyrifos. Similar to the time course, there was considerably more cholinesterase inhibition of maternal brain activity than fetal brain activity. The time course data in A and B, as well as age-matched control data, have appeared previously in Lassiter et al. (1998).
likely, some of the brain TCP is generated through cholinesterase inhibition, an interpretation that is supported by the fact that both cholinesterase inhibition and TCP levels peak at the same time. There may be other factors at play, such as the disposition of TCP into the fetal brain from the circulation, which would be dependent on the physicochemical environment of the maternal and fetal brain. At this age, the fetal brain has much less lipid and more water content than does the maternal brain (Benjamins and McKhann, 1981). This fact, however, would seem to argue against the partition of TCP from the blood preferentially into the fetal brain, as TCP is a lipophilic compound and practically insoluble in water (Mitsu
Industries Ltd, www.mitsuindia.com). One way to define experimentally the TCP profile of distribution would be to inject TCP into the pregnant dam and follow the distribution and time course. It is curious that the dam liver contains so much more TCP than the fetal liver, but the pattern is reversed for the brain. Possibly the liver acts as a much more efficient detoxifier in the dam than in the fetus. That is, in the dam, even though the initial concentration of chlorpyrifos is higher, the maternal liver more readily detoxifies chlorpyrifos because of its strong complement of P450s, A-esterases, and carboxylesterases before chlorpyrifos or its metabolites reach the brain, resulting in
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a 20- to 30-fold difference in the concentration of TCP between the dam liver and dam brain. In the fetus, however, there is no difference in TCP concentration in the liver or brain tissue; this pattern leads us to propose the hypothesis that the fetal liver does not play a significant role in detoxification and, therefore, TCP (and most likely also chlorpyrifos and chlorpyrifos-oxon) just distributes evenly throughout the fetal compartment. There are other instances in the literature where the distribution of a compound given to the pregnant mother reaches higher concentrations in the fetus (as reviewed by Dencker, 1982; see also Salama et al., 1993) or specifically the fetal brain compared to the maternal brain: carbaryl (Strother and Wheeler, 1980); diphenylhydantoin (Mirkin, 1973); dihydromorphine (Sanner and Woods, 1965); phencyclidine (Ahmad et al., 1987); or D 9-THC (Mirkin, 1973). Moreover, it is also interesting to note that, in the present study, the fetal/maternal difference in brain TCP concentration did not decrease as the chlorpyrifos dosage decreased: at a dosage of either 3 or 7 mg/kg, the fetal brain TCP concentration was approximately threefold that of the maternal brain. Taken in concert, these results and those of our previous studies (Lassiter et al., 1998) indicate that during late gestation the fetus is just as accessible, if not more accessible, as the dam to orally administered chlorpyrifos. These results raise concerns regarding the presence of chlorpyrifos, its metabolites, or cholinesterase inhibition in the fetal compartment. ACKNOWLEDGMENTS
opment: New findings and toxicological implications. Environ. Health Perspect. 107, 59 – 64. Brzak, K. A., Harms, D. W., Bartels, M. J., and Nolan, R. J. (1998). Determination of chlorpyrifos, chlorpyrifos-oxon, and 3,5,6-trichloro-2-pyridinol in rat and human blood. J. Anal. Toxicol. 22, 203–210. Chanda, S. M., Harp, P., Liu, J., and Pope, C. N. (1995). Comparative developmental and maternal neurotoxicity following acute gestational exposure to chlorpyrifos in rats. J. Toxicol. Environ. Health 44, 189 –202. Chanda, S. M., and Pope, C. N. (1996). Neurochemical and neurobehavioral effects of repeated gestational exposure to chlorpyrifos in maternal and developing rats. Pharmacol. Biochem. Behav. 53, 771–776. Claborn, H. V., Hoffman, R. A., Mann, H. D., and Oehler, D. D. (1968). Residues of Dursban and its oxygen analog in the body tissues of treated cattle. J. Econ. Entomol. 61, 983–986. Cochran, R. C., Kishiyama, J., Aldous, C., Carr, W. C., Jr., and Pfiefer, K. F. (1995). Chlorpyrifos: Hazard assessment based on a review of the effects of short-term and long-term exposure in animals and humans. Food Chem. Toxicol. 33, 165–172. Dencker, L. (1982). Disposition of chemicals in the developing embryo and fetus. Int. J. Biol. Res. Pregnancy 3, 114 –121. Dishburger, H. J., McKellar, R. L., Pennington, J. Y., and Rice, J. R. (1977). Determinations of residues of chlorpyrifos, its oxygen analogue and 3,5,6trichloro-2-pyridinol in tissues of cattle fed chlorpyrifos. J. Agric. Food Chem. 25, 1325–1328. Drevenkar, V., Vasilic, Z., Stengl, B., Frobe, Z., and Rumenjak, V. (1993). Chlorpyrifos metabolites in serum and urine of poisoned persons. Chem.– Biol. Interact. 87, 315–322. Dutton, G. J. (1978). Developmental aspects of drug conjugation, with special reference to glucuronidation. Annu. Rev. Pharmacol. Toxicol. 18, 17–35. Dutton, G. J., and Leakey, J. E. (1981). The perinatal development of drugmetabolizing enzymes: What factors trigger their onset? Prog. Drug Res. 25, 189 –273.
We thank Drs. S. Barone, Jr., S. C. Bondy, S. M. Chanda, S. McMaster, and L. G. Sultatos for thoughtful critique of earlier drafts of this manuscript. We also thank Dr. S. Barone, Jr., Dr. Sushmita M. Chanda, Kay Riggsbee, and especially Rene´e Marshall for assisting in this study. The authors also express our gratitude for the assistance of Dennis House in the statistical analysis of this data. Chlorpyrifos was generously supplied by Dow AgroSciences LLC. T. L. Lassiter is supported by NIEHS Training Grant T32 ES07126. This research has been reviewed by the National Health and Ecological Environmental Effects Research Laboratory, U.S. EPA, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor mention of trade names or commercial products constitute endorsement or recommendation for use.
Dvorchik, B. H., Woodward, G., Sitar, D. S., and Tweed, W. A. (1986). Hydroxylation and glucuronidation of various xenobiotics by hepatic microsomes from the fetal lamb, pregnant ewe and human fetus. Dev. Pharmacol. Ther. 9, 282–289.
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Fukuto, T. R. (1990). Mechanism of action of organophosphorus and carbamate insecticides. Environ. Health Perspect. 87, 245–254.
Ahmad, G., Halsall, L. C., and Bondy, S. C. (1987). Persistence of phencyclidine in fetal brain. Brain Res. 415, 194 –196.
Hunter, D. L., Marshall, R. S., and Padilla, S. (1997). Automated instrument analysis of cholinesterase activity in tissues from carbamate-treated animals: A cautionary note. Toxicol. Meth. 7, 43–53.
Aspelin, A. L. (1997). Pesticide Industry Sales and Usage: 1994 and 1995 Market Estimates. Office of Pesticide Programs, U. S. Environmental Protection Agency, EPA 733-R-97-002, Washington, D. C. Barron, M. G., Plakas, S. M., and Wilga, P. C. (1991). Chlorpyrifos pharmacokinetics and metabolism following intravascular and dietary administration in channel catfish. Toxicol. Appl. Pharamacol. 108, 474 – 482. Benjamins, J. A., and McKhann, G. M. (1981). Development, regeneration, and aging of the brain. In Basic Neurochemistry, 3rd ed. (G. J. Siegal, R. W. Albers, B. W. Agranoff, and R. Katzman, Eds.), pp. 445– 469. Little, Brown and Company, Boston. Brimijoin, S., and Koenigsberger, C. (1999). Cholinesterases in neural devel-
Ecobichon, D. J. (1996). Toxic effects of pesticides. In Casarett and Doull’s Toxicology, The Basic Science of Poisons, 5th ed. (C. D. Klaassen, Ed.), pp. 643– 689. McGraw-Hill, New York. Ellman, G. L., Courtney, D. K., Andres, V., and Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88 –95. Eto, M. (1974). Organophosphorus Pesticides: Organic and Biological Chemistry. CRC Press, Cleveland, OH.
Ivey, M. C., Mann, H. D., Oehler, D. D., Claborn, H. V., Eschile, J. L., and Hogan, B. F. (1972). Chlorpyrifos and its oxygen analogue: Resides in the body tissues of dipped cattle. J. Econ. Entomol. 65, 1647–1649. Ivey, M. C., and Palmer, J. S. (1979). Chlorpyrifos and 3,5,6-trichloro-2pyridinol: Residues in body tissues of swine treated with chlorpyrifos for hog louse and itch mite control. J. Econ. Entomol. 72, 837– 838. Ivey, M. C., Palmer, J. S., and Hooten, E. C. (1978). Chlorpyrifos and 3,5,6-trichloro-2-pyridinol: Residues in the body tissues of cattle wearing chlorpyrifos-impregnated plastic ear bands. J. Econ. Entomol. 71, 697–700. Johnson, C. D., and Russell, R. L. (1975). A rapid, simple radiometric assay for
TOXICOKINETICS OF CHLORPYRIFOS IN THE PREGNANT RAT cholinesterase, suitable for multiple determinations. Anal. Biochem. 64, 229 –238. Juchau, M. R., Chao, S. T., and Omiecinski, C. J. (1980). Drug metabolism by the human fetus. Clin. Pharmacokinet. 5, 320 –339. Karczmar, A. G., Srinivasan, R., and Bernsohn, J. (1973). Cholinergic function in the developing fetus. In Fetal Pharmacology (L. Bore´us, Ed.), pp. 127–177. Raven Press, New York. Lassiter, T. L., Padilla, S., Mortensen, S. R., Chanda, S. M., Moser, V. C., and Barone, S., Jr. (1998). Gestational exposure to chlorpyrifos: Apparent protection of the fetus? Toxicol. Appl. Pharmacol. 152, 56 – 65. Layer, P. G., and Willbold, E. (1995). Novel functions of cholinesterase in development, physiology, and disease. Prog. Histochem. Cytochem. 29, 1–94. Lucier, G. W. (1976). Perinatal development of conjugative enzyme systems. Environ. Health Perspect. 18, 25–34. Mann, H. D., Ivey, M. C., Kunz, S. E., and Hogan, B. F. (1973). Chlorpyrifos, its oxygen analogue and 3,5,6-trichloro-2-pyridinol: Residues in body tissues of turkeys confined in pens on treated soil. J. Econ. Entomol. 66, 715–717. Martinasevic, M. K., King, C. D., Rios, G. R., and Tephly, T. R. (1998). Immunohistochemical localization of UDP-glucuronosyltransferases in rat brain during early development. Drug Metab. Dispos. 26, 1039 –1041. McKellar, R. L., Dishburger, H. J., Rice, J. R., Craig, L. F., and Pennington, J. (1976). Residues of chlorpyrifos, its oxygen analogue, and 3,5,6-trichloro2-pyridinol in milk and cream from cows fed chlorpyrifos. J. Agric. Food Chem. 24, 283–286. Michalek, H., Pintor, A., Fortuna, S., and Bisso, G. M. (1985). Effects of diisopropylfluorophosphate on brain cholinergic systems of rats at early developmental stages. Fundam. Appl. Toxicol. 5, S204 –S212. Mirkin, B. L. (1973). Drug distribution in pregnancy. In Fetal Pharmacology (L. Bore´us, Ed.), pp. 1–27. Raven Press, New York. Moser, V. C., Chanda, S. M., Mortensen, S. R., and Padilla, S. (1998). Ageand gender-related differences in sensitivity to chlorpyrifos in the rat reflect developmental profiles of esterase activities. Toxicol. Sci. 46, 211–222. Moser, V. C., and Padilla, S. (1998). Age- and gender-related differences in the time course of behavioral and biochemical effects produced by oral chlorpyrifos in rats. Toxicol. Appl. Pharmacol. 149, 107–119. Nolan, R. J., Rick, D. L., Freshour, N. L., and Saunders, J. H. (1984). Chlorpyrifos: Pharmacokinetics in human volunteers. Toxicol. Appl. Pharmacol. 73, 8 –15.
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Pope, C. N., Charkraborti, T. K., Chapman, M. L., Farrar, J. D., and Arthun, D. (1991). Comparison of the in vivo cholinesterase inhibition in neonatal and adult rats by three organophosphorothioate insecticides. Toxicology 68, 51– 61. Reinke, L. A., Belinsky, S. A., Kauffman, F. C., Evans, R. K., and Thurman, R. G. (1982). Regulation of NADPH-dependent mixed-function oxidation in perfused livers. Comparative studies with sorbitol and ethanol. Biochem. Pharmacol. 31, 1621–1628. Salama, A. K., Bakry, N. M., and Abou-Donia, M. B. (1993). A review article on placental transfer of pesticides. J. Occup. Med. Toxicol. 2, 383–397. Sanner, J. H., and Wood, L. S. (1965). Comparative distribution of tritiumlabeled dihydromorphine between maternal and fetal rats. J. Pharmacol. Exp. Ther. 148, 176 –184. Santhoshkumar, P., and Shivanandappa, T. (1994). Differential in vivo inhibition of the foetal and maternal brain acetylcholinesterase by bromophos in the rat. Neurotoxicol. Teratol. 16, 227–232. SAS Institute (1989). SAS/STAT User’s Guide, Version 6, 4th ed, Vol. 2, Cary, NC. Small, D. H., Michaelson, S., and Sberna, G. (1996). Non-classical actions of cholinesterases: Role in cellular differentiation, tumorigenesis, and Alzheimer’s disease. Neurochem. Int. 28, 453– 483. Smith, G. N., Watson, B. S., and Fischer, F. S. (1967). Investigations on Dursban insecticide. Metabolism of [ 36Cl]O,O-diethyl O-3,5,6-trichloro-2pyridyl phosphorothioate in rats. J. Agric. Food Chem. 15, 132–138. Strother, A., and Wheeler, L. (1980). Excretion and disposition of [ 14C]carbaryl in pregnant, non-pregnant and foetal tissues of the rat after acute administration. Xenobiotica 10, 113–124. Suleman, F. G., Abid, A., Gradinaru, D., Daval, J. L., Magdalou, J., and Minn, A. (1998). Identification of the uridine diphosphate glucuronosyltransferase isoform UGT1A6 in rat brain and in primary cultures of neurons and astrocytes. Arch. Biochem. Biophys. 358, 63– 67. Suleman, F. G., Ghersi-Egea, J. F., Leininger-Muller, B., and Minn, A. (1993). Uridine diphosphate-glucuronosyltransferase activities in rat brain microsomes. Neurosci. Lett. 161, 219 –222. Sultatos, L. G., Costa, L. G., and Murphy, S. D. (1982). Determination of organophosphorus insecticides, their oxygen analogs and metabolites by high pressure liquid chromatography. Chromatographia 15, 669 – 671. Sultatos, L. G., Minor, L. D., and Murphy, S. D. (1985). Metabolic activation of phosphorothioate pesticides: Role of the liver. J. Pharmacol. Exp. Ther. 232, 624 – 628.
Gestational Exposure to Chlorpyrifos: Comparative Distribution of Trichloropyridinol in the Fetus and Dam D. L. Hunter,* ,1 T. L. Lassiter,* ,† and S. Padilla* *Cellular and Molecular Toxicology Branch, Neurotoxicology Division, National Health Effects and Ecological Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711; and †Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina Received January 26, 1999; accepted April 14, 1999
signs and symptoms characteristic of overstimulation of the cholinergic nervous system, presumably because of accumulation of the neurotransmitter acetylcholine in the synaptic cleft (as reviewed by Fukuto, 1990; Ecobichon, 1996). In the developing nervous system, acetylcholine, acetylcholinesterase, and butyrylcholinesterase (EC 3.1.1.8), all molecules that are likely to be affected by anticholinesterases, are postulated to play developmental roles that are unique from their ultimate function in the adult nervous system (as reviewed by Karczmar et al., 1973; Layer and Willbold, 1995; Small et al., 1996; Brimijoin and Koenigsberger, 1999). Consequently, basic research is needed to characterize the distribution of an anticholinesterase insecticide to determine if that insecticide actually reaches the fetal nervous system during brain development and to compare the concentration measured in the fetal brain to the level in the maternal brain. Chlorpyrifos (Dursban, Lorsban, O,O9-diethyl O-[3,5,6trichloro-2-pyridyl] phosphorothionate) is a widely used organophosphorus insecticide, both in agricultural and non-agricultural arenas (Cochran et al., 1995; Aspelin, 1997). Previous studies in our laboratory as well as others have shown that neonatal rats are much more sensitive to the acute effects of this insecticide than are adults (Pope et al., 1991; Moser and Padilla, 1998; Moser et al., 1998). Previous investigators believed that the fetus was not as affected by gestational exposure to anticholinesterases because the prenatal rat has the mother’s physiology to “protect” it from toxicity (Michalek et al., 1985; Santhoshkumar and Shivanandappa, 1994; Chanda et al., 1995; Chanda and Pope, 1996). In our studies of the prenatal rat, however, we have found that the fetal compartment is not protected from cholinesterase inhibition when the pregnant dam is dosed orally with chlorpyrifos. In fact, the fetal brain shows as much cholinesterase inhibition after one oral dose of chlorpyrifos to the dam as does the maternal brain (Lassiter et al., 1998). One shortcoming of our previous study was the lack of toxicokinetic data on the distribution of chlorpyrifos and its metabolites. This information is integral to gaining a better understanding of the toxicodynamic and toxicokinetic parameters describing chlorpyrifos intoxication of the pregnant rat and fetus.
Gestational Exposure to Chlorpyrifos: Comparative Distribution of Trichloropyridinol in the Fetus and Dam. Hunter, D. L., Lassiter, T. L., and Padilla, S. (1999). Toxicol. Appl. Pharmacol. 158, 16 –23. Chlorpyrifos (O,O*-diethyl O-[3,5,6-trichloro-2-pyridyl] phosphorothionate) is a commonly used anticholinesterase insecticide, and therefore the potential for human exposure is high. The present time course and dose response studies were conducted to delineate the toxicokinetics of chlorpyrifos and its metabolites in the pregnant rat and fetus. Time-pregnant, Long–Evans rats were treated orally with chlorpyrifos during late gestation (Gestational Days 14 –18). Following euthanasia the level of chlorpyrifos and its metabolites, chlorpyrifos-oxon and 3,5,6-trichloro-2-pyridinol (TCP), were measured in both fetal and maternal brain and liver (limits of quantitation: 59.2, 28.8, and 14.0 ng/g tissue, respectively). In addition, cholinesterase inhibition was also measured in the same tissues for comparison. TCP was the only component detected. The highest level of TCP and the lowest level of cholinesterase activity showed the same time of peak effect: 5 h after the last dose. The concentration of TCP in the maternal liver was approximately fivefold higher than the TCP concentration in fetal liver, but, paradoxically, the concentration of TCP in the fetal brain was two- to fourfold higher than the TCP concentration in the maternal brain. The half-life of the TCP was identical in all tissues examined (12–15 h). These toxicokinetic results suggest that the fetal nervous system may be exposed to a higher concentration of chlorpyrifos than the maternal nervous system when the dam is orally exposed to chlorpyrifos during late gestation. Key Words: fetus; chlorpyrifos; chlorpyrifos-oxon; trichloropyridinol; pregnancy.
Anticholinesterase insecticides are thought to mediate toxicity primarily through inhibition of an esterase, acetylcholinesterase (EC 3.1.1.2). The inhibition of this enzyme leads to 1 To whom correspondence should be addressed: Cellular and Molecular Toxicology Branch, Neurotoxicology Division, Mail Drop 74-B, National Health Effects and Ecological Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711. Fax: (919) 541-5024; E-mail: [email protected].
16 0041-008X/99
TOXICOKINETICS OF CHLORPYRIFOS IN THE PREGNANT RAT
The present group of studies was conducted to assess and compare the distribution of chlorpyrifos and its metabolites in pregnant rats dosed with chlorpyrifos. Some of the dosing scenarios used in this group of studies replicate those employed in the previous study (i.e., Lassiter et al., 1998). The current studies were designed to monitor the concentration of chlorpyrifos and its metabolites in critical tissues in the fetus and dam. In brief, pregnant dams were orally dosed with chlorpyrifos for 5 days during late gestation, and the amounts of chlorpyrifos, chlorpyrifos-oxon (the potent anticholinesterase metabolite), and 3,5,6-trichloro-2-pyridinol (TCP, 2 the “leaving group”, see Eto, 1974) were quantified in fetal and maternal brain and liver; cholinesterase inhibition was monitored in the same tissues for comparison. Both time course and dose response assessments were conducted. The results support and extend our original observation that the fetal nervous system is not protected from chlorpyrifos toxicity and, in fact, more of this insecticide may be distributed to the fetal nervous system than to the maternal nervous system. MATERIALS AND METHODS Animals, Treatment, and Sample Collection Experimental design. Time-pregnant, primiparous Long–Evans rats were obtained from Charles River Laboratories Crl: (LE) BR (Portage, ME) and were determined to be sperm-positive on Gestational Day 0 (GD0). Dams arrived on GD4 and were housed individually on a 12:12 h light:dark cycle, with food (Rodent Diet 5001; Lab Diet, Indianapolis, IN) and water provided ad libitum. Weight gain was monitored from arrival to euthanasia. Dams were distributed among groups on GD13 according to a ranking of body weight gain to ensure balanced body weights among groups. Following chlorpyrifos exposure, weight gain also was used as a general index of maternal health status. For the time course study, groups of dams received 0 or 7 mg/kg of chlorpyrifos (dose volume: 1 ml/kg) on GD14 –18 and were euthanized at 2, 5, 10 (GD18), 24 (GD19), or 48 (GD20) h after the last dose (n/dose/time point 5 4). Additional dams and pups were euthanized at 120 h (Postnatal Day 1, P1) after the last dose. The day of birth was considered P0. Similarly, the dams in the dose response study were dosed on GD14 –18 by gavage with 0, 3, or 7 mg/kg chlorpyrifos in corn oil (n 5 4/dose group). All dose response animals, however, were euthanized at 5 h after the last dose (time of peak concentration of TCP). Sample collection. Tissue collection and analysis were the same for time course and dose response portions of this study. All dams and pups were anesthetized with CO 2 and euthanized by decapitation for collection of tissue samples. The dams were perfused transcardially with saline. The perfusion continued until the normally dark liver was a very pale brown. At each time point dam liver and brain were collected and frozen on dry ice. Half of the brain was used for chromatography measurements and half was used for assay of cholinesterase activity. Separate liver samples were collected as well. The uterus was removed, placed on ice, and the position of any resorptions recorded. Sampling roughly every other fetus in the uterus, a total of eight fetuses was removed. From each litter, two fetal livers and brains were collected individually for cholinesterase assays while the other six sets were pooled for HPLC analysis. All samples were immediately frozen on dry ice. Tissues were stored at 280°C until analysis. Litter was considered the smallest
2
Abbreviations used: ANOVA, analysis of variance; GD, gestational day; LOQ, limits of quantitation; P, postnatal day; TCP, 3,5,6-trichloro-2-pyridinol.
17
unit of analysis, meaning that end points recorded for individual fetuses from a single litter were averaged as replicates. Toxicokinetic Analysis Sample preparation. All tissues for HPLC analysis were diluted using 0.1 M Na phosphate buffer (pH 7.4) and homogenized on ice (Setting 6, Polytron, Brinkman Industries, Westbury, NY). All tissues were diluted 1:3 (w/v: e.g., 1 g of tissue in 2 ml of buffer), with the exception of fetal brain, which was diluted 1:1.05. Tissue homogenates were extracted using a method based on Sultatos et al. (1982). One milliliter of tissue homogenate was added to a tube containing 1 g of NaCl; chlorpyrifos-methyl was added as an internal standard. The extractant ethyl acetate (4 ml) was added next and the samples were then vortexed for 5 min (Setting 7, multitube vortexer, Dade International, Miami, FL). The samples were then centrifuged for 10 min at 1000g (HNS-centrifuge, Damon, Needham Heights, MA). After the ethyl acetate layer was transferred to a collection tube, a second volume of ethyl acetate was added to the pellet and a second extraction was performed. Following addition of the second ethyl acetate layer to the collection tube, a gentle stream of nitrogen gas was used to evaporate the ethyl acetate. The fully dried extracts were reconstituted in 200 ml of the tissue-appropriate mobile phase. Chromatography. Analyses were made using a system manufactured by Waters (Milford, MA) consisting of a 717plus Autosampler, a 600 Solvent Delivery System, and a 996 PDA detector. The column used was a mBondapak C18 column (300 mm long 3 3.9 mm id). The mobile phase appropriate for brain samples was 71.5% methanol, 28.5% HPLC-grade water, with the pH adjusted to 3.4 using glacial acetic acid. For analysis of liver the mobile phase was 60% acetonitrile, 40% HPLC-grade water (pH 3.4). The volume of sample injected was 50 ml, the flow rate was 1.5 ml/min, and the wavelength was 290 nm. A calibration curve was established using TCP, chlorpyrifos-oxon, and chlorpyrifos standards, ranging in concentration from 5 to 500 ng, which were dissolved in the appropriate mobile phase. Spiking of control samples revealed that TCP and chlorpyrifos-oxon were recovered at 95% and recovery of chlorpyrifos was 80%. The limits of quantitation (LOQ) were set at 10 times the baseline noise. The LOQ were 14, 29, and 59 ng/g of tissue for TCP, chlorpyrifos-oxon, and chlorpyrifos, respectively. The components of interest were quantified using peak heights. A representative chromatogram of prepared standard solutions of chlorpyrifos and its metabolites is presented in Fig. 1. There was concern that some TCP may have been conjugated to glucuronide by Phase II detoxification systems. Therefore, prior to analyzing any samples, a Glusulase control experiment was performed based on a method described by Reinke et al. (1982). Homogenates of liver from chlorpyrifos-treated dams (1 ml, 1:3) were incubated with and without 10 ml of Glusulase (900 U of glucuronidase, 135 U of sulfatase, New England Nuclear, Boston, MA) for 90 min at room temperature. The samples were then extracted as described previously. The amount of TCP quantified in the Glusulase-treated and parallel control samples was the same (Fig. 2). This control experiment established that Glusulase treatment was not necessary in the preparation of the experimental samples. Biochemical Analysis Sample homogenization. Samples were homogenized (larger samples, . 50 mg wet wt: 20 s, Setting 6, Polytron) or sonicated on ice (smaller samples, , 50 mg wet wt: 10 s, Setting 3, Branson Sonifier 250) in 0.1 M Na phosphate buffer (pH 8) containing 1% Triton X-100. Maternal liver was homogenized for 45 s due to the high collagen content. Activity for the chlorpyrifos-treated subjects was expressed as percent inhibition. Cholinesterase activity. Total cholinesterase activity (acetylcholinesterase and butyrylcholinesterase) was measured for dam brain and fetal brain using an Hitachi 911 Automatic Analyzer (Boehringer Mannheim Corp., Indianapolis, IN) according to the method outlined in Hunter et al. (1997). The 911 is a spectrophotometer-equipped robot that dispenses sample, buffer, chromogen, and substrate to assay cholinesterase activity according to a variation of the
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HUNTER, LASSITER, AND PADILLA
FIG. 1. Representive chromatogram of chlorpyrifos and metabolites. The peaks are A, TCP; B, chlorpyrifos-oxon; C, chlorpyrifos-methyl (internal standard); and D, chlorpyrifos.
Ellman et al. (1961) method. Considering the high sulfhydryl background present in the liver and resultant reaction with the chromogen used in the Ellman assay, it was prohibitive to use this colorimetric technique. Fetal and maternal liver cholinesterase activity, therefore, was assayed using the Johnson and Russell (1975) radiometric assay with a final substrate concentration of 1.2 mM. The [ 3H]acetate produced in the hydrolysis of [ 3H]acetylcholine was quantified using a Wallac 1410 liquid scintillation counter.
Statistics In the time course study, the cholinesterase inhibition and TCP concentration were analyzed the same way. A two-way analysis of variance (ANOVA) (treatment vs time) was performed on each of the four tissues (fetal and maternal brain and liver). Step-down, one-way ANOVAs were then used to determine the treatment-related effects across the time course (SAS Institute, 1989). In the dose response study, the statistical analysis of cholinesterase inhibition data and the TCP concentration data was the same. For each of the four tissues (fetal and maternal brain and liver), a one-way ANOVA was used to determine if there were statistical differences among any of the treatment groups. In all cases, the dose effect was significant. Tukey’s multiple comparison procedure was used to determine which dose groups were different. The T21 of TCP was calculated based on a straight line fitted to the log-transformed TCP data for the 5, 10, 24, and 48 h time points.
RESULTS
Glusulase Treatment
FIG. 2. Glusulase treatment. Parallel sets of samples were prepared with or without Glusulase treatment. Glusulase treatment was intended to liberate TCP that had been conjugated to glucuronide. The concentration of TCP in maternal liver was unaffected by pretreatment with Glusulase.
To determine if glucuronide conjugates of TCP had been formed, liver tissue from chlorpyrifos-treated dams was incubated with Glusulase, which would hydrolyze the conjugates and produce free TCP, which could be measured. A corresponding aliquot of the same liver homogenate that was not treated with Glusulase was extracted concurrently. The levels of TCP were measured in both samples and the results compared in Fig. 2. There was no difference in TCP concentrations
TOXICOKINETICS OF CHLORPYRIFOS IN THE PREGNANT RAT
19
whether the sample was treated with Glusulase, which indicated that glucuronide-conjugates of TCP were not present in adult rat liver. Maternal liver was chosen because, among the tissues sampled, it is the one most likely to glucuronidate TCP (Lucier, 1976; Dutton, 1978; Juchau et al., 1980; Dutton and Leakey, 1981; Dvorchik et al., 1986; Suleman et al., 1993, 1998; Martinasevic et al., 1998). Because the Glusulase treatment had no effect on maternal liver TCP concentrations, Glusulase treatment of the other samples was not necessary. Overt Toxicity No overt maternal toxicity was noted in either the time course or dose response studies. The rate of weight gain for controls and chlorpyrifos-treated dams was comparable (data not shown). Furthermore, gestational chlorpyrifos treatment did not affect fetal body/brain weights, number of resorptions, or litter size (data not shown). Toxicokinetic Analysis In the time course study, pregnant rats were dosed with chlorpyrifos (0 or 7 mg/kg/day) on GD14 –18 and euthanized at various intervals after the last dose. Livers collected from dams and fetuses were analyzed for the presence of chlorpyrifos and its metabolites using HPLC. The only analyte present in measurable quantities was TCP. The TCP concentration time course indicated that, while maximal concentrations of TCP were measured 5 h after the last dose for both maternal and fetal liver, the maternal liver contained fivefold more TCP than the fetal liver at this time point (Fig. 3A). TCP levels decreased sharply after the 5-h time point in both maternal and fetal liver and were below the LOQ 48 h postdosing in the fetus and by P1 in the dams. The analysis of brain tissue from the time course study produced some unexpected results. The fetal brain TCP concentration was twice as high as the maternal brain concentration 5 h after the last dose of chlorpyrifos (the peak of TCP concentration; Fig. 3B). TCP levels decreased sharply after the 5-h time point in both the dam and fetal brain, with no measurable TCP in either tissue 48 h after the last dose of chlorpyrifos. In the dose response study, pregnant rats were treated with chlorpyrifos (0, 3, or 7 mg/kg/day) on GD14 –18 and euthanized 5 h after the last dose. As in the time course study, maternal and fetal brain and liver were analyzed for chlorpyrifos and its metabolites. Again, TCP was the only metabolite present in measurable quantities, with maternal liver concentrations much higher than fetal liver levels for both the 3 and 7 mg/kg/day treatments: 8.0- and 6.6-fold, respectively (Fig. 4A). For both the maternal and fetal liver, the 7 mg/kg/day treatment resulted in higher TCP levels than the 3 mg/kg/day treatment (Fig. 4A). For the brain, the unusual results revealed in the time course study were replicated in the dose response study. TCP levels
FIG. 3. Time course of trichloropyridinol in the liver and brain. (A) TCP levels in the liver were highest 2–10 h after the last 7 mg/kg gestational dose of chlorpyrifos. The amount of TCP in the maternal liver was elevated above the fetal liver amount for at least 48 h after the last dose of chlorpyrifos. }, Statistically significant increase in TCP between the fetal and maternal tissue, at that particular time point. The areas under the curve were 6164 and 32,147 ng 3 day/g of tissue for fetal and maternal liver, respectively. The T21 of TCP in the fetal and maternal liver was comparable after repeated, gestational exposure: 15.4 and 14.0 h, respectively. The LOQ is indicated by the dotted horizontal line. (B) Similar to TCP levels in the liver, TCP in the fetal and maternal brain were highest 2–10 h after the last gestational dose of chlorpyrifos. Most importantly, at 2 and 5 h after the last dose there was more than twice as much TCP in the fetal brain as in the maternal brain. The areas under the curve were 2783 and 1,768 ng 3 day/g of tissue for fetal and maternal brain, respectively. T21 of TCP was 12.5 and 14.8 h for fetal and maternal brain, respectively.
were much higher in the fetal brain than in the maternal brain (225 vs 76 ng/g in the 7 mg/kg/day group and 123 vs 36 ng/g in the 3 mg/kg/day group; Fig. 4B). In the fetal brain, the 7 mg/kg/day treatment caused a higher concentration of TCP than the 3 mg/kg dosage level. The amount of TCP in the maternal brain was comparable at both dosage levels (Fig. 4B). Cholinesterase Inhibition Cholinesterase activity levels were determined in liver and brain from maternal and fetal rats in both the time course and
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dose of chlorpyrifos, but the maternal brain cholinesterase activity was still inhibited 44% on P1, 5 days after the last dose. The results of the time course study were replicated in the dose response, as there was more cholinesterase inhibition in maternal liver than in fetal liver (3.5- and 1.5-fold, 3 and 7 mg/kg/day treatments, respectively; Fig. 5C). Cholinesterase inhibition was similar for both chlorpyrifos treatments in the maternal liver (84 vs 89%, 3 vs 7 mg/kg/day, respectively), but the fetal liver cholinesterase activity was more inhibited by the 7 mg/kg/day treatment than the lower treatment level (Fig. 5C). The dose response results for brain tissue indicated that there was also much more cholinesterase inhibition in the maternal brain than in the fetal brain: 87 vs 32% in the 7 mg/kg/day group and 41 vs 3% in the 3 mg/kg/day group (Fig. 5D). In both maternal and fetal brain, the 7 mg/kg/day treatment resulted in more cholinesterase inhibition than the 3 mg/kg/day dosage of chlorpyrifos (Fig. 5D). DISCUSSION
FIG. 4. Dose response of trichloropyridinol in the liver and brain. (A) Similar to the time course, there was more TCP in the maternal liver than fetal liver at both dosage levels. The 7 mg/kg dosage of chlorpyrifos caused higher levels of TCP in the maternal and fetal liver than the 3 mg/kg dosage. *Statistically significant increase in TCP at the higher dose; in the same tissue, }, statistically significant difference in TCP concentration between the fetal and maternal tissue, at that particular dose. The LOQ is indicated by the dotted horizontal line. (B) Following repeated, gestational chlorpyrifos exposure, there was more TCP in the fetal brain than the maternal brain: 4.5- and 3.0-fold more at 3 and 7 mg/kg chlorpyrifos, respectively. Repeated exposure to 7 mg/kg chlorpyrifos also resulted in more TCP in the fetal brain than exposure to the 3 mg/kg dosage level. The amount of TCP in the maternal brain was comparable at both dosages. When the levels are compared across tissue, the maternal liver has more TCP than the maternal brain at both 3 and 7 mg/kg, but this is not true for the fetal tissues: the amount of TCP in the fetal brain is comparable to the amount in the fetal liver at 3 or 7 mg/kg.
dose response studies. In the time course study the time span of maximal cholinesterase inhibition in all tissues was 5–10 h after the last gestational dose of chlorpyrifos. Cholinesterase inhibition recovered after the 5–10-h time points with fetal levels returning to normal sooner than dam levels. Maternal liver cholinesterase was twofold more inhibited than fetal liver activity (Fig. 5A). Similarly, the maternal brain exhibited more cholinesterase inhibition than fetal brain at 5 h after the last dose of chlorpyrifos (84 vs 25%; Fig. 5B). Fetal brain cholinesterase recovered to control levels by 24 h after the last
The results of the present group of studies imply that the fetus is not protected from chlorpyrifos when the dam is dosed orally; these toxicokinetic results parallel the toxicodynamic results reported in a previous group of experiments (Lassiter et al., 1998). In fact, in the present study, the fetal brain received a higher dose of TCP than did the maternal brain. These higher levels of TCP were not a result of increased accumulation within the fetal compartment, i.e., that the TCP was created in and was unable to leave the fetal compartment. If that were the case, the T12 of TCP in the fetal tissues would be much longer than in the maternal tissues, but the T21s for TCP in maternal and fetal tissues were not different. The calculated T21 for maternal or fetal brain or liver was 12–15 h, which agrees well with previously published data for rat liver (Smith et al., 1967). There are three logical explanations as to why only TCP was detected in the liver and brain: (1) TCP had the lowest limit of quantitation; (2) chlorpyrifos-oxon was not detected because it is an extremely ephemeral molecule that is rarely detected in tissues of animals treated in vivo with chlorpyrifos (e.g., Ivey et al., 1972; Mann et al., 1973; McKellar et al., 1976; Barron et al., 1991; Drevenkar et al., 1993), except fat (Claborn et al., 1968), or even after in vitro “spiking” of tissues (Ivey et al., 1972; Dishburger et al., 1977; Brzak et al., 1998); and (3) chlorpyrifos is rarely detected in any tissue except blood (Drevenkar et al., 1993; Nolan et al., 1984) or fatty tissue (Claborn et al., 1968; McKellar et al., 1976; Ivey et al., 1978; Ivey and Palmer, 1979) and usually after a relatively high dose exposure. The TCP could have arisen from either a detoxification (e.g., hydrolysis by P450s or A-esterases, or binding to carboxylesterases; Sultatos et al., 1985) or toxic reaction (e.g., binding of chlorpyrifos-oxon to cholinesterase). Because of the low levels of detoxification enzymes in the brain tissue, TCP in the brain probably arose from the binding of the oxon to cholinesterase or through TCP in the circulation. More than
TOXICOKINETICS OF CHLORPYRIFOS IN THE PREGNANT RAT
21
FIG. 5. Time course and dose response of cholinesterase inhibition in the liver and brain. (A) Fetal liver cholinesterase was maximally inhibited 2–5 h after the last dose of chlorpyrifos (43% inhibition), while maternal liver cholinesterase was more than 80% inhibited at 2, 5, and 10 h after the last treatment. There was recovery of fetal liver cholinesterase activity by 10 h after the last dose, but cholinesterase in the maternal liver did not recover until 48 h after the last chlorpyrifos exposure. }, Statistically significant increase in cholinesterase inhibition between the fetal and maternal tissue, at that particular dose. (B) Maximal cholinesterase inhibition occurred at 5–10 h after the last dose of chlorpyrifos. There was, however, 4.7-fold more inhibition in the maternal brain than in the fetal brain. Fetal brain cholinesterase inhibition recovered by 24 h after the last dose, but maternal brain activity did not recover completely within the days sampled (GD18 –P1). (C) Fetal liver cholinesterase activity demonstrated dose-dependent inhibition at 3 and 7 mg/kg. *Statistically significant increase in cholinesterase inhibition at the higher dose, in the same tissue. Maternal liver cholinesterase activity was maximally inhibited at both dosage levels. (D) Fetal brain cholinesterase activity was not inhibited at the lower dose, but gestational exposure to the 7 mg/kg dosage did cause inhibition. Maternal brain cholinesterase was inhibited in a dose dependent fashion at 3 and 7 mg/kg chlorpyrifos. Similar to the time course, there was considerably more cholinesterase inhibition of maternal brain activity than fetal brain activity. The time course data in A and B, as well as age-matched control data, have appeared previously in Lassiter et al. (1998).
likely, some of the brain TCP is generated through cholinesterase inhibition, an interpretation that is supported by the fact that both cholinesterase inhibition and TCP levels peak at the same time. There may be other factors at play, such as the disposition of TCP into the fetal brain from the circulation, which would be dependent on the physicochemical environment of the maternal and fetal brain. At this age, the fetal brain has much less lipid and more water content than does the maternal brain (Benjamins and McKhann, 1981). This fact, however, would seem to argue against the partition of TCP from the blood preferentially into the fetal brain, as TCP is a lipophilic compound and practically insoluble in water (Mitsu
Industries Ltd, www.mitsuindia.com). One way to define experimentally the TCP profile of distribution would be to inject TCP into the pregnant dam and follow the distribution and time course. It is curious that the dam liver contains so much more TCP than the fetal liver, but the pattern is reversed for the brain. Possibly the liver acts as a much more efficient detoxifier in the dam than in the fetus. That is, in the dam, even though the initial concentration of chlorpyrifos is higher, the maternal liver more readily detoxifies chlorpyrifos because of its strong complement of P450s, A-esterases, and carboxylesterases before chlorpyrifos or its metabolites reach the brain, resulting in
22
HUNTER, LASSITER, AND PADILLA
a 20- to 30-fold difference in the concentration of TCP between the dam liver and dam brain. In the fetus, however, there is no difference in TCP concentration in the liver or brain tissue; this pattern leads us to propose the hypothesis that the fetal liver does not play a significant role in detoxification and, therefore, TCP (and most likely also chlorpyrifos and chlorpyrifos-oxon) just distributes evenly throughout the fetal compartment. There are other instances in the literature where the distribution of a compound given to the pregnant mother reaches higher concentrations in the fetus (as reviewed by Dencker, 1982; see also Salama et al., 1993) or specifically the fetal brain compared to the maternal brain: carbaryl (Strother and Wheeler, 1980); diphenylhydantoin (Mirkin, 1973); dihydromorphine (Sanner and Woods, 1965); phencyclidine (Ahmad et al., 1987); or D 9-THC (Mirkin, 1973). Moreover, it is also interesting to note that, in the present study, the fetal/maternal difference in brain TCP concentration did not decrease as the chlorpyrifos dosage decreased: at a dosage of either 3 or 7 mg/kg, the fetal brain TCP concentration was approximately threefold that of the maternal brain. Taken in concert, these results and those of our previous studies (Lassiter et al., 1998) indicate that during late gestation the fetus is just as accessible, if not more accessible, as the dam to orally administered chlorpyrifos. These results raise concerns regarding the presence of chlorpyrifos, its metabolites, or cholinesterase inhibition in the fetal compartment. ACKNOWLEDGMENTS
opment: New findings and toxicological implications. Environ. Health Perspect. 107, 59 – 64. Brzak, K. A., Harms, D. W., Bartels, M. J., and Nolan, R. J. (1998). Determination of chlorpyrifos, chlorpyrifos-oxon, and 3,5,6-trichloro-2-pyridinol in rat and human blood. J. Anal. Toxicol. 22, 203–210. Chanda, S. M., Harp, P., Liu, J., and Pope, C. N. (1995). Comparative developmental and maternal neurotoxicity following acute gestational exposure to chlorpyrifos in rats. J. Toxicol. Environ. Health 44, 189 –202. Chanda, S. M., and Pope, C. N. (1996). Neurochemical and neurobehavioral effects of repeated gestational exposure to chlorpyrifos in maternal and developing rats. Pharmacol. Biochem. Behav. 53, 771–776. Claborn, H. V., Hoffman, R. A., Mann, H. D., and Oehler, D. D. (1968). Residues of Dursban and its oxygen analog in the body tissues of treated cattle. J. Econ. Entomol. 61, 983–986. Cochran, R. C., Kishiyama, J., Aldous, C., Carr, W. C., Jr., and Pfiefer, K. F. (1995). Chlorpyrifos: Hazard assessment based on a review of the effects of short-term and long-term exposure in animals and humans. Food Chem. Toxicol. 33, 165–172. Dencker, L. (1982). Disposition of chemicals in the developing embryo and fetus. Int. J. Biol. Res. Pregnancy 3, 114 –121. Dishburger, H. J., McKellar, R. L., Pennington, J. Y., and Rice, J. R. (1977). Determinations of residues of chlorpyrifos, its oxygen analogue and 3,5,6trichloro-2-pyridinol in tissues of cattle fed chlorpyrifos. J. Agric. Food Chem. 25, 1325–1328. Drevenkar, V., Vasilic, Z., Stengl, B., Frobe, Z., and Rumenjak, V. (1993). Chlorpyrifos metabolites in serum and urine of poisoned persons. Chem.– Biol. Interact. 87, 315–322. Dutton, G. J. (1978). Developmental aspects of drug conjugation, with special reference to glucuronidation. Annu. Rev. Pharmacol. Toxicol. 18, 17–35. Dutton, G. J., and Leakey, J. E. (1981). The perinatal development of drugmetabolizing enzymes: What factors trigger their onset? Prog. Drug Res. 25, 189 –273.
We thank Drs. S. Barone, Jr., S. C. Bondy, S. M. Chanda, S. McMaster, and L. G. Sultatos for thoughtful critique of earlier drafts of this manuscript. We also thank Dr. S. Barone, Jr., Dr. Sushmita M. Chanda, Kay Riggsbee, and especially Rene´e Marshall for assisting in this study. The authors also express our gratitude for the assistance of Dennis House in the statistical analysis of this data. Chlorpyrifos was generously supplied by Dow AgroSciences LLC. T. L. Lassiter is supported by NIEHS Training Grant T32 ES07126. This research has been reviewed by the National Health and Ecological Environmental Effects Research Laboratory, U.S. EPA, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor mention of trade names or commercial products constitute endorsement or recommendation for use.
Dvorchik, B. H., Woodward, G., Sitar, D. S., and Tweed, W. A. (1986). Hydroxylation and glucuronidation of various xenobiotics by hepatic microsomes from the fetal lamb, pregnant ewe and human fetus. Dev. Pharmacol. Ther. 9, 282–289.
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