Age- and Gender-Related Differences in Sensitivity to Chlorpyrifos in the Rat Reflect Developmental Profiles of Esterase Activities

TOXICOLOGICAL SCIENCES ARTICLE NO.

46, 211–222 (1998)

TX982526

Age- and Gender-Related Differences in Sensitivity to Chlorpyrifos in the Rat Reflect Developmental Profiles of Esterase Activities Virginia C. Moser, Sushmita M. Chanda,1 Spencer R. Mortensen,2 and Stephanie Padilla National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711 Received March 13, 1998; accepted June 22, 1998

Age- and Gender-Related Differences in Sensitivity to Chlorpyrifos in the Rat Reflect Developmental Profiles of Esterase Activities. Moser, V. C., Chanda, S. M., Mortensen, S. R., and Padilla, S. (1998). Toxicol. Sci. 46, 211–222. Young rats are more sensitive than adults to a single oral dose of chlorpyrifos, an organophosphorus pesticide. A direct comparison of chlorpyrifos effects in young (postnatal day 17; PND17), adolescent (PND27), and adult (70 days) Long–Evans rats was conducted to determine quantitative and possibly qualitative differences in sensitivity in terms of behavioral changes and cholinesterase (ChE; total cholinesterase activity) inhibition at these three ages. Male and female rats were administered chlorpyrifos orally at one of two doses (PND17, 5 or 20 mg/kg; PND27, 20 or 50 mg/kg; adult, 20 or 80 mg/kg) and tested at either 3.5 or 6.5 h after dosing. Behavioral testing included observational evaluations and measurements of motor activity and was followed immediately by tissue collection for ChE determination in brain and blood. For both behavioral changes and ChE inhibition, peak effects occurred at 3.5 h in adult male and PND27 rats (both sexes) and at 6.5 h in adult female and PND17 rats (both sexes). Comparisons of the 20 mg/kg dose across ages showed generally less ChE inhibition and fewer behavioral effects with increasing age, except that the adult females were similar to the PND27 rats. The high dose used for each age group produced similar brain ChE inhibition (80 –90%) and generally similar behavioral effects. Interestingly, a few endpoints in the young rats were less affected than in adults at this level of ChE inhibition. The degree of ChE inhibition in the brain more closely paralleled the blood inhibition in the younger rats, compared to the adults. Carboxylesterase (CaE) and A-esterase are known to play an important role in the detoxification of organophosphates and may be partially responsible for these sensitivity differences. Liver and plasma CaE and A-esterase activities were measured in untreated male rats on PND1, 4, 7, 12, 17, and 21 and in adults of both sexes (82–92 days old). Preweanling rats had considerably less activity of both enzymes, and adult females had less liver CaE activity than males. These differences in detoxifying enzymes correlate with the age-related differences in behavioral and biochemical effects, as well as the gender dif1 Supported by cooperative agreement No. CT901915 with the University of North Carolina at Chapel Hill. Current address: Sugen Co., Redwood City, CA. 2 Supported by cooperative agreement No. CT902813 with Clemson University. Current address: American Cyanamid Co., Agricultural Products Research Division, Princeton, NJ.

ferences seen in adult rats, and thus may be a major influence on the differential sensitivity to chlorpyrifos.

In general, young animals appear to be more sensitive to the lethal effects of organophosphorus (OP) pesticides compared to adults (see for example, Brodeur and DuBois, 1963; Gaines and Linder, 1986; Harbison, 1975; Lu et al., 1965), but few studies have addressed toxic effects at sublethal doses (National Research Council, 1993). Chlorpyrifos (O,O9-diethyl O9-[3,5,6-trichloro-2-pyridinol]phosphorothionate) is a widely used OP that, when bioactivated to chlorpyrifos-oxon, exerts toxicity through inhibition of the enzyme acetylcholinesterase (AChE; EC 3.1.1.7). Young animals are more sensitive to the neurotoxicity induced by chlorpyrifos than adults: for example, in a recent study, preweanling Long–Evans rats were approximately five to seven times more sensitive than adult rats to the behavioral and biochemical effects precipitated by orally administered chlorpyrifos (Moser and Padilla, 1998). Several other studies using different routes of administration have also reported similar greater sensitivity to chlorpyrifos in younger rats (Atterberry et al., 1997; Pope and Chakraborti, 1992; Pope et al. 1991; Whitney et al., 1995). The mechanism(s) for the greater susceptibility of young rats is not well defined. Studies have shown that an acute oral dose of chlorpyrifos produces signs typical of cholinergic overstimulation, such as hypothermia, salivation, lacrimation, diarrhea, incoordination, tremors, and fasciculations; altered motor function was one of the most sensitive endpoints evaluated in these studies (Mattsson et al., 1996; Moser, 1995; Nostrandt et al., 1997). Comparisons of 17-day-old and adult rats revealed similar neurobehavioral effects, albeit at lower doses in the younger rats (Moser and Padilla, 1998). That study also reported some other differences: (1) the recovery of cholinesterase inhibition was faster in the young rats and (2) in adult but not young rats, gender-related differences were observed in terms of the magnitude and time course of chlorpyrifos effects. Carboxylesterase (CaE; EC 3.1.1.1) and A-esterase (EC 3.1.8.1) are known to play an important role in the detoxification of OPs (Jokanovic´ et al., 1996; Maxwell, 1992b; Reiner, 1993; Walker, 1993). OPs bind irreversibly to the esteratic site

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of CaE in a stoichiometric manner (Maxwell, 1992b), and this reaction therefore may reduce the effective concentration of chlorpyrifos-oxon at the target enzyme site (AChE). In contrast to the CaE, A-esterases are Ca21-dependent enzymes which hydrolyze OPs in a nonstoichiometric manner to form nontoxic metabolites, without concomitant inhibition (Aldridge, 1953a,b). The A-esterase responsible for the hydrolysis of chlorpyrifos-oxon to its less-toxic metabolites, 3,5,6-trichloro2-pyridynol and diethylphosphate, is known as chlorpyrifosoxonase (CPFO-ase; Furlong et al., 1989; Mortensen et al., 1996). It has been suggested that for some OPs, kinetic factors play a greater role in age-dependent susceptibility than do other factors, such as differences in the target enzyme (Benke and Murphy, 1975; Brodeur and DuBois, 1967; Sterri et al., 1985). The greater sensitivity observed in the young animals could be due to lower levels of CaE, A-esterase, or other OP-detoxifying enzymes. The design of this experiment allowed direct comparisons of chlorpyrifos toxicity in several ways. First, the effects of the same chlorpyrifos dose (20 mg/kg) could be compared, using both behavioral and biochemical endpoints, across three age groups. The inclusion of two chlorpyrifos doses allowed a preliminary assessment of the slope of the dose–response curves. Differences between males and females, both within the same age group and across ages, could also be evaluated. In addition, since previous studies indicate a difference in the time of peak effect between postnatal day 17 (PND17) pups and adults (Moser and Padilla, 1998), this study incorporated a comparison of the two time points to verify that earlier finding. Finally, these results can be compared to the control activity of CaE and A-esterase at various ages. METHODS Chemicals Chlorpyrifos was purchased from Chem Service (Westchester, PA, .99% pure). Chlorpyrifos-oxon was obtained from the U.S. EPA Repository Research Triangle Park, NC (.98% purity). 3,5,6-Trichloro-2-pyridinol (TCP) was a gift from Dr. Carey Pope (Northeast Louisiana University, Monroe, LA). All other chemicals used were analytical grade. For the in vivo study, chlorpyrifos was suspended in corn oil (Mazola) and administered by oral gavage at 2 ml/kg. Animals Long–Evans hooded rats [CRL:(LE)BR] were obtained from Charles River Laboratories Inc. (Kingston, NY, for behavioral studies and adult esterase data and Portage, MI, for esterase ontogeny), housed on heat-treated pine shavings or hardwood chip bedding, and allowed food (Purina Rodent Chow No. 5001) and water ad libitum. The AAALAC-accredited animal facility was maintained at 72 6 2°C, 50 6 10% humidity, with a 12-h light:dark cycle. All animal handling procedures were followed as outlined by the NIH guidelines and approved by the U.S. EPA Animal Care and Use Committee. For studies requiring young rats, pregnant female Long–Evans rats were obtained on specified gestational days and allowed to deliver naturally. On PND4 (day of birth, PND0), rat pups were randomly culled to litters with five males and five females. Rat pups were housed with the dams until weaning on

PND21, after which time they were separated by sex and housed five/cage until the end of the study. Adult rats were received from the supplier at 60 days of age and housed individually. The behavioral study employed rats (n 5 5/dose/time/sex/age) at ages of 17, 27, and approximately 70 days (to be exact, 68, 70, or 75 days). For the developmental profiles of CaE and A-esterase, plasma and liver tissues were collected from male rats on PND1, 4, 7, 12, 17, 21, and 92 (n 5 3/age; drawn from separate litters). Adult male and female control esterase data were determined on tissues from rats 68 – 80 days (CaE; n 5 5/sex) or 115 days old (A-esterase; n 5 6/sex). Experimental Design Behavioral and biochemical studies. All three age groups (17, 27, and 70 days) were represented in each days’ testing, and although testing was staggered, the dose, sex, and age groups were counterbalanced across days. Rats of each age were dosed with one of three doses, depending on age: PND17, vehicle (corn oil), 5, or 20 mg/kg; PND27, vehicle, 20, or 50 mg/kg; and adult, vehicle, 20, or 80 mg/kg. Note that all ages received a dose of 20 mg/kg, in order to make direct comparisons across ages using the same dose. Half of each age/dose group was tested at 3.5 h after the dose, and the other half was tested at 6.5 hours. Immediately after testing, rats were euthanized and whole blood and brain were taken for cholinesterase measurements. Esterase profiles. Developmental profiles were determined for the activity of carboxylesterase and A-esterase in untreated rats. For these ontogeny studies, plasma and liver tissues were collected from male rats 1 to 92 days of age. These tissues were assayed all at one time. In addition, CaE and A-esterase data for male and female adult rats were considered for evaluation of gender differences. These data were obtained in the course of other studies (Chanda et al., 1997; Mortensen, 1997). Behavioral Testing Selected endpoints of the functional observational battery (FOB) as well as measurements of motor activity were used to evaluate behavioral and neurological changes (McDaniel and Moser, 1993; Moser et al., 1988). The endpoints included those measures consistently altered by exposure to cholinesterase inhibitors (as detailed in Moser, 1995), with modifications (e.g., small changes in scoring criteria) as necessary for testing preweanling rats (Moser and Padilla, 1998). Behavioral testing required 3– 4 min per rat and was followed by a 30-min session in the motor activity chambers. Upon removal from the activity chamber, the rat was euthanized for tissue collection. On test days, rats were removed from the animal holding rooms and transported to an isolated laboratory. At the specified time, the observer removed the test subject from the cage and held it briefly. Lacrimation, salivation, and handling reactivity were scored according to defined criteria. The rat was then placed on a laboratory cart top (60 3 90 cm) with a perimeter barrier (6.5 cm high) covered with a clean absorbent pad. The rat was observed moving about on the cart undisturbed for 2 min, during which time the observer evaluated and scored any gait abnormalities, ataxia (PND27 and adults only), arousal, and level of activity. In addition, tremor and clonic movements, if present, were recorded. The rat’s responses to an auditory click stimulus (metal clicker) and to a tail pinch (using forceps) were also scored. In PND27 and adult rats, the ability of the pupil to constrict in response to light was then assessed. Aerial righting ability was evaluated in PND27 and adult rats, whereas the PND17 pups were placed on their backs and the ability to attain normal posture was ranked. Rectal temperature data were collected in PND27 and adult rats, and body weight was measured in all rats. The same observer conducted all tests and was unaware of the rat’s treatment level. Motor activity data were collected immediately after FOB testing, using a maze composed of a series of interconnected alleys shaped like a figure-eight with two blind alleys projecting from the central arena (Reiter, 1983). Six pairs of phototransmitter diodes were equally spaced around the figure-eight portion of the maze, and one pair was located in each of the blind alleys (total of eight

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DEVELOPMENT OF CHLORPYRIFOS SENSITIVITY detectors). Activity, recorded by a microprocessor, was based on the number of photocell interruptions during the 30-min session. Tissue Collection Immediately after behavioral evaluations, rats were euthanized by decapitation under CO2-induced anesthesia. Trunk blood was collected in heparinized tubes and the brain was removed. For esterase studies in untreated rats, trunk blood was collected in heparinized tubes and separated into plasma and erythrocytes by low-speed centrifugation (2500g for 5 min). Liver was perfused with approximately 120 ml of saline at room temperature, and a sample was taken (from the same site in all rats). All tissues were stored at 280°C until time of assay. Carboxylesterase Assay Carboxylesterase activity was determined as detailed in Chanda et al. (1997). Briefly, tissues were diluted with Tris buffer (20 mM, pH 8.0) using 20 ml (PND1–12) or 10 ml (PND17–92) of liver homogenate 1:1000 (w/v), using 10 ml of 1:10 (PND92) or 1:5 (PND21) plasma, or 5 ml of undiluted plasma (PND1–17). The substrate p-nitrophenyl acetate was then added. Ethylene glycol bis (b-aminoethyl ether) N,N,N9,N9-tetraacetic acid (EGTA, 1 mM final concentration) was added to the plasma and liver samples to inhibit Ca21dependent hydrolases (i.e., A-esterases). The increase in absorbance at 405 nm was read using a Thermomax microtiter plate reader (Molecular Devices Inc., Menlo Park, CA) which maintained temperature at 26°C. CaE activity was calculated as nmoles of p-nitrophenol produced/minute. Cholinesterase Assay Cholinesterase (ChE) activity was determined using a Hitachi 911 automatic analyzer (Boehringer Mannheim Corp., Indianapolis, IN), as previously described (Hunter et al., 1997; Willig et al., 1996). The Hitachi 911 is a spectrophotometer which uses robotics for dispensing reagents, etc.; the assay method is based on that of Ellman et al. (1961). Brain tissue was homogenized 1:50, and whole blood was diluted 1:10, using 0.1 M sodium phosphate buffer (pH 8.0) with 1% Triton X-100. A subset of tissues was assayed using the microtiter plate-based method (Nostrandt et al., 1993) to determine conversion factors for use in determining actual ChE activity based on the 911 units. ChE activity is expressed as micromoles acetylthiocholine hydrolyzed/min/gram of wet tissue weight or milliliter of blood. Because both AChE and butyrylcholinesterase (BuChE) hydrolyze acetylthiocholine, this assay does not differentiate the two esterases. Chlorpyrifos-oxonase (A-esterase) Assay CPFO-ase activity was determined based on the method of Furlong and co-workers (1989) with modifications, described in detail by Mortensen and co-workers (1996). Briefly, tissues for this assay were prepared as follows: plasma samples from young rats (PND1, 4, 7, and 12) were used undiluted, PND17 and 21 plasma samples were diluted 1:5 (v/v) (1 ml plasma added to 4 ml of 0.1 M Tris–HCl buffer, pH 8.5), and adult plasma samples were diluted 1:10. Perfused liver samples for all ages were homogenized 1:10 (w/v) (1 g perfused liver tissue added to 9 ml 0.1 M Tris–HCl buffer, pH 8.5, containing 1% Triton X-100). The total reaction volume of 1 ml contained 930 ml 0.1 M Tris–HCl buffer, pH 8.5, 50 ml of a 40 mM CaCl2 solution, and 10 ml liver homogenate or plasma. The reaction was initiated by the addition of 10 ml of chlorpyrifos-oxon (1 mM final concentration) in methanol; methanol alone was used as a blank. The change in optical density at 310 nm (due to the generation of TCP as chlorpyrifos-oxon is hydrolyzed by CPFO-ase) was measured every 20 s for 6 min at 30°C using a Beckman DU-70 spectrophotometer. Statistics Behavioral test measures were analyzed as previously described (see Creason, 1989; Moser et al., 1988). Initially, two-way analyses of variance (ANO-

VAs) were conducted for data within each age group. When significant overall effects were obtained, analyses at each time point were conducted to determine which dose groups were different from the control for that time point (i.e., concurrent controls). In addition, three-way ANOVAs were conducted using between-subject factors of age, dose, and time. Continuous data were analyzed by a general linear model (SAS, 1990). Rank-order data were analyzed using a categorical modeling procedure (CATMOD; SAS, 1990) which fits linear models to functions of response frequencies and then by weighted regression. In all cases, resulting probability values ,0.05 were considered significant. ChE data were treated in a manner similar to that employed for the continuous behavioral data. Liver and plasma A-esterase and CaE activities were compared between ages at different time points by one-way ANOVA, followed by Tukey’s post hoc test of multiple comparisons.

RESULTS

Comparing the effects of the same dose at each age is a direct method of evaluating the age-related differences in sensitivity to chlorpyrifos. Figure 1 presents brain ChE activity following either vehicle or 20 mg/kg chlorpyrifos, at both time points and in both sexes. Control ChE activity showed an age-related increase in both males and females. There were no differences in control ChE activity between the two time points (3.5 and 6.5 h) for any age group. Following a single dose of chlorpyrifos, brain ChE was inhibited in all age groups, with the magnitude of inhibition greatest in the youngest rats (PND17). Statistical analyses of the data at this dose showed that there was an overall effect of age for both males and females. These age differences were similar at both time points in male rats (age-by-time factor not significant), whereas in females the age differences were dependent on the time of assessment (age-by-time interaction p , 0.0001). The age differences were evident in both the absolute ChE activity and the inhibition relative to the age-matched controls. This was most apparent in males, wherein inhibition was about five-fold greater in PND17 pups compared to adults at both time points (87% inhibition vs 39% at 3.5 h, 89% vs. 43% at 6.5 h). PND27 rats showed about twice as much inhibition as adults (69% inhibition vs 39%, and 70% vs. 43%, at 3.5 and 6.5 h, respectively). Female rats showed a more complicated pattern than did males. At 3.5 h after dosing, the relationship between brain ChE inhibition and age was apparent, although the magnitude of the differences was less than in males (about three-fold, 87% inhibition vs 66% for PND17 vs adults). On the other hand, at 6.5 h the adult females showed greater inhibition, i.e., were more sensitive, than were the PND27 rats. At that time point, PND17 pups were about three-fold more affected than the PND27 rats (91% inhibition vs 69%), but only twice as affected as adults (91 vs 78%). Comparisons of the sexes within each age group showed that the data for PND17 and PND27 males and females were quite similar, whereas adult female brain ChE was inhibited to a greater extent than was adult male ChE. Thus, the data for this dose of chlorpyrifos reveal that the age-related differences in brain ChE inhibition depend on the sex of the subject as well as the time of assessment.

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FIG. 1. Comparison of brain ChE activity following a single dose of 20 mg/kg chlorpyrifos in male and female rats ages 17, 27, and 70 days, at 3.5 and 6.5 h after dosing. ChE activity is shown as micromoles of substrate hydrolyzed/minute/gram tissue. Numbers over the chlorpyrifos bars indicate percentage of appropriate control values (age and time matched).

Blood ChE was greatly inhibited following 20 mg/kg chlorpyrifos in all age groups (88 –96% inhibition; see Fig. 2). Although the range of differences was quite small, analyses revealed a significant overall effect of age; i.e., the inhibition varied with age. As with the brain ChE data, only in female rats did the correlations between inhibition and age vary with the time of evaluation (age-by-time interaction p , 0.034). In addition to comparing the effects of the same dose across ages, this study was designed to provide a range of effective doses for each age group. Figure 2 shows the effects of both doses for rats of each age, at both times and in both sexes. The highest dose in each age group (PND17, 20 mg/kg; PND27, 50 mg/kg; adults, 80 mg/kg) produced similar (i.e., not statistically different) brain and blood ChE inhibition at each time point. At the time of maximal effects, brain ChE inhibition

averaged, for males and females, respectively: 92 and 93% inhibition at PND17; 86 and 88% inhibition at PND27; 83 and 90% inhibition in adults. Thus, in terms of ChE inhibition, these high doses were equieffective. Since the blood and brain ChE activities were evaluated in the same rats, we can determine the relationship of inhibition between the two compartments. Figure 3 represents brain ChE activity plotted as a function of blood ChE activity (both as percentage of control). In the rat pups, brain inhibition closely matches blood inhibition, and the slope of the regression line approaches one (slopes, 1.75 and 1.72 for males and females, respectively). In the PND27 rats, the line becomes steeper (slopes, 2.55 and 2.41), and in adult males, steeper still (slope, 5.03). In contrast to the adult males, the adult female (slope, 2.28) and PND27 data overlapped. In other words, the degree of ChE inhibition in the two compartments was similar in PND17 pups, but as the age of the rat increased, considerably more blood inhibition was present compared to brain ChE. Table 1 lists the measures of the behavioral test battery and doses which were significantly different from control at each time point. Chlorpyrifos produced qualitatively similar effects in all age groups. That is, signs of toxicity included gait changes, tremors, smacking, depressed activity/reactivity levels (in the open field and motor activity chambers), lacrimation, antinociception, and hypothermia (not measured in PND17 rats). Interestingly, righting was impaired in PND17 rats and adult females, but not PND27 or adult males. Measures that are not listed in Table 1 (handling reactivity, click response, and salivation) were not altered by chlorpyrifos. The behavioral signs of toxicity produced by chlorpyrifos (20 mg/kg) followed the same pattern of age-related sensitivity described above for ChE inhibition data. This dose produced a full syndrome of cholinergic stimulation in PND17 pups, only a few effects in PND27 rats, and even fewer effects in adult males. Temporal differences in effects (between 3.5 and 6.5 h) were more striking in the behavioral data than in the data on ChE inhibition. To illustrate this, Fig. 4 presents the ranking of tremor. In male rats of all ages, the lowest dose used for each age did not produce any type of tremorigenic activity. The high dose at 3.5 h, however, produced a similar degree of tremor in each age group (rating scales for pups and postweanling rats are slightly different, but the maximum score indicates severe tremors in both). At 6.5 h, the magnitude of these tremors increased in the PND17 pups (upward shift of the curve shown on Fig. 4), whereas it lessened in the PND27 and adult rats (downward shifts). Data collected from females were similar in that the low dose either produced no tremors or else caused mild tremors in two of the five rats tested (PND27 at 20 mg/kg). At 6.5 h, a greater effect was seen in female PND17 rats, and less effect was seen in PND27 rats: the same pattern observed in male rats. In contrast to the adult males, however, adult females showed considerably more effect at 6.5 h (upward shift), at which time the tremors were more severe than those seen in the PND27 group or in males. Thus, as was

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FIG. 2. ChE activity in blood and brain following both doses of chlorpyrifos in each age, at both time points, expressed as percentage of appropriate control values. Brain ChE control values are presented in Fig. 1; blood control values were PND17, males 1.64 6 0.15, females 1.58 6 0.13; PND27, males 1.42 6 0.13, females 1.40 6 0.10; adult, males 1.50 6 0.09, females 1.59 6 0.07 (mean 6 SD; mmol substrate hydrolyzed/min/ml blood).

evident in the ChE data, the pattern of the age-related differences depends on the gender of the test subject as well as the time of assessment. The time-course characteristics described for tremors (Fig. 4) were evident in most other behavioral endpoints as well. In general, there were differences in group means between the two test times, and in some cases these temporal differences attained statistical significance (i.e., significant dose-by-time interaction; these are indicated in Table 1). This occurred most frequently in adults, with a larger effect at 3.5 h in males and at 6.5 h in females. Although the dose-by-time interactions were not as frequently significant in younger rats, PND17 rats generally showed more effects at 6.5 h, and PND27 at 3.5 h, in both sexes. Since behavioral data and ChE inhibition were collected in the same subjects, we sought to examine whether the signs of chlorpyrifos-induced toxicity occurred at similar levels of ChE inhibition. Thus, the relationship between ChE inhibition and behavioral changes observed were examined for each endpoint. In general, similar signs were observed at similar levels of inhibition; however, some exceptions were noted. Motor activity levels, as measured in the figure-eight mazes, were differently affected. This is illustrated in Fig. 5, which shows motor activity levels plotted as a function of the level of brain

ChE inhibition in each dose/age group. At the dose producing 80 –90% brain ChE inhibition, the magnitude of motor activity depression was greater in the PND27 and adult rats (about 90% decrease in activity counts), compared to PND17 pups (40 – 60% decrease); this was the case for both sexes. To a lesser extent, the magnitude of open-field activity depression, decreased tail-pinch responsiveness, and occurrence of mouth smacking was greater in the postweanling rats. For example, following the high dose, mouth smacking was observed in three of five male PND17 pups, four of five PND27 rats, and in all male adult rats. Liver and plasma samples collected from control rats of different ages (PND1–92) were assayed for CPFO-ase as well as CaE activity. The developmental curves of these two enzymes are shown in Fig. 6. Shortly after weaning, CPFO-ase activity had achieved adult levels, but CaE activity was still about one-half that of adults. Figure 6 also provides a comparison of adult male and female esterase activities. Female adult rats had about one-half the liver CaE activity as males, while plasma CaE activities were the same. In contrast, there were no gender differences in liver CPFO-ase activities, while a small (30%) but significantly greater activity in plasma samples from females was observed.

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FIG. 3. ChE activity in brain plotted as a function of blood ChE activity, both expressed as percentage of the average control data. Each point represents each treated male or female rat; data from both doses and both time points (3.5 and 6.5 h) are included. Dashed lines indicate linear regressions fit to the data for each age group (PND17, PND27, or adult).

DISCUSSION

A gradual decrease in sensitivity to chlorpyrifos toxicity as the rat matures is a consistent finding, in this laboratory and by others (Atterberry et al., 1997; Moser and Padilla, 1998; Pope et al., 1991; Whitney et al., 1995). While an earlier study in our laboratory (Moser and Padilla, 1998) documented the time course of effects following a maximally tolerated dose (MTD) in adults and PND17 rats, the present study extends our knowledge of chlorpyrifos by providing, in three age groups and both sexes, the following information: (1) a direct comparison of behavioral and biochemical effects following the same dose; (2) a preliminary dose–response assessment using doses less than the MTD; and (3) evaluation of the time of peak effect

after oral dosing. We also provide the ontogeny of esterase activities in control rats in order to compare these kinetic factors with the developmental pattern of sensitivity to chlorpyrifos. Comparisons of a single oral dose of chlorpyrifos (20 mg/ kg) in males revealed differences in sensitivity that were approximately fivefold for PND17 rats, and twofold for PND27 rats, compared to adults. In female rats, comparisons at 20 mg/kg chlorpyrifos also revealed age-related differences, but the magnitude of the differences was not as great as in males since the adult females were more similar in sensitivity to the PND27 rats. The preliminary dose–response curves for both behavioral and biochemical effects were generally shifted to the left in the younger rats. The time of assessment was a confounding factor; brain ChE inhibition and behavioral alterations were significantly greater at the later time point in adult female, but not male, rats. The influence of time of assessment in adult female rats was reported in our previous time-course study (Moser and Padilla, 1998), and that finding was verified and extended to PND27 rats in the present study. If only the later time point (6.5 h) had been used to compare responses in different age groups, the conclusions would be quite different, because at that time the adult females actually showed greater ChE inhibition and more behavioral changes than did PND27 rats. These data suggest, therefore, that characterization of age-related differences requires a knowledge of the influence of gender of the test subject as well as the time of assessment. A possible explanation for age-related differences in toxicity is a change in the relationship between the amount of ChE inhibition and subsequent behavioral changes. In earlier studies, we reported that considerable brain ChE inhibition ($50%) in adult and PND17 rats was attained before behavioral effects were evident (Moser and Padilla, 1998; Nostrandt et al., 1997). In the present study, this relationship was also evident. In the PND17 rats, the low dose produced 50 – 60% brain ChE inhibition (apparent “threshold”), and the only significant change observed was decreased open-field arousal (females only). Individual rats with slightly higher ChE inhibition showed additional signs (smacking and depressed activity and tail-pinch response). Adult male brain ChE was inhibited 40 –50%, and there was a clear correlation between ChE inhibition and motor activity; the rats with more inhibition showed lower motor activity counts. For adult females, and PND27 rats of both sexes, the low dose produced .60% inhibition as well as numerous behavioral changes. Thus, prediction of behavioral effects based on a threshold level of brain ChE inhibition (40 –50% inhibition following an acute dose) appears reasonable for each age group. The high dose of chlorpyrifos produced substantial ChE inhibition as well as marked neurobehavioral signs in all age groups. While the magnitude of these changes was similar across ages, there were a few exceptions. Preweanling rats showed less decreased motor activity, less depression of the

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TABLE 1 Measures of the Neurobehavioral Battery Altered by Chlorpyrifos and the Doses Which Were Significantly Different from Control, for Males and Females of Each Age Group PND17 Function/endpoint

3.5 h

PND27 6.5 h

Adult

3.5 h

6.5 h

3.5 h

6.5 h

Males Neuromuscular/vestibular Gait changes Righting deficits Convulsive Tremors Smacking Activity/reactivity 2Motor activity 2Open field activity 2Arousal Autonomic Lacrimation Other 2Tail-pinch response 2Body temperature

20 20

20 20

50 —

— —

80 —

80 —

20 20

20 20

50 50

— —

80 80a

80 —

20 — 20

20 20 20

20, 50 50 50a

20, 50 20, 50 —

20, 80 80a 80a

80 — —

20

20

50

—

80a

—

20 ND

— ND

50 20, 50

50 50

80 80

— —

Females Neuromuscular/vestibular Gait changes Righting deficits Convulsive Tremors Smacking Activity/reactivity 2Motor activity 2Open field activity 2Arousal Autonomic Lacrimation Other 2Tail-pinch response 2Body temperature

20 20

20a 20

50 —

50 —

80 —

20, 80a 80

20 20

20 20

50 50

— —

80 80

80a 80

20 — 20

20 — 5, 20a

20, 50a 50 50

50 20, 50 20, 50

80 80 80

20, 80a 20, 80 20, 80a

20

20

50

—

80

80

20 ND

20 ND

20, 50a 50

50 50

80 80

20, 80a 20, 80

Note. ND, not determined in PND17 rats. a Significantly more effect than at other time point.

tail-pinch response, and less lacrimation and smacking. Antinociception, lacrimation, and mouth movements have been shown to be mediated through central muscarinic receptors (e.g., Iwamoto and Marion, 1993; Kelley et al., 1989; Koehn and Karczmar, 1978; Pedigo et al., 1975; Salamone et al., 1986, 1990), whereas motor activity is an apical measure which can be altered by either muscarinic or nicotinic agents. Since preweanling rats have fewer muscarinic binding sites in at least some brain areas (e.g., Moser and Padilla, 1998; Tice et al., 1996; Zhu et al., 1996), it is possible that stimulation of the muscarinic system could produce less effects than in the matured systems. This finding that mature rats showed greater CNS depression (decreased activity levels) and more muscarinic receptor-mediated effects (antinociception, smacking, lac-

rimation) differs from human case reports comparing anticholinesterase poisonings in young and older humans (Lifshitz et al., 1997; Sofer et al., 1989). In those reports, children present more CNS depression (coma, stupor) than do adults, and nicotinic signs such as fasciculations are rarely observed in the young. These case histories, however, report mostly poisonings by carbamates, with only a few involving OP pesticides. Comparing blood and brain ChE inhibition in the different age groups provides information regarding: (1) the usefulness of blood ChE inhibition for predicting brain ChE inhibition following acute exposure and (2) the ability of the blood to detoxify chlorpyrifos oxon. In this case, the relationship between ChE inhibition in the two compartments was different for the three age groups. Thus, predictions for brain ChE inhibition based on blood data would

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AChE to BuChE in blood differs with age; if chlorpyrifos differentially inhibits one over the other, this could influence the results shown in Fig. 3. While we could locate nothing in the literature regarding the ratio of these two enzymes during development, our preliminary data indicate that male rat plasma is approximately 40% BuChE from PND5 on. It is known that hepatic P450 enzymes are important in both the activation and the deactivation of chlorpyrifos and other OPs (e.g., Chambers et al., 1994; Sultatos et al., 1984). Young and female rats have less active liver metabolism than adult males (Imaoka et al., 1991; Kato, 1974; MacLeod et al., 1972), which could partially explain the increased OP susceptibility. Others have investigated this hypothesis and have shown that the activation to toxic oxons is lower in young and female rats, which would lead to lowered, not greater, sensitivity (Atterberry et al., 1997; Benke and Murphy, 1975; Ma and Chambers, 1994; Murphy and DuBois, 1958; Sultatos, 1991). Thus, microsomal enzyme-mediated activation/deactivation probably

FIG. 4. Scoring of tremors (mean score) in rats of each age as a function of chlorpyrifos dose administered (two doses for each age group). Filled symbols indicate data at 3.5 h after dosing; open symbols, 6.5 h. Tremors are scored on a scale from 1 (no tremors) to either 3 (for PND17 pups) or 4 (for postweanling rats), which indicates severe tremors (modified scales were necessary for differences in normal manifestation of tremors between adult and preweanling rats).

produce erroneous conclusions. In adults, blood ChE was almost completely inhibited before brain ChE was altered; this was also reported in Nostrandt et al. (1997). In younger rats, there was a more direct correlation between these compartments; i.e., the degree of inhibition more closely reflects the degree of brain inhibition (Fig. 3). Such a scenario could indicate either: (1) an immature blood–brain barrier, which would allow chlorpyrifos (or its oxon) to equilibrate into the brain more easily, or (2) the presence of a detoxifying system in the blood, which becomes more efficient as the rat matures, and removes some of the compound before it reaches the brain. It is known that the blood– brain barrier is not fully mature at 17 days of age in rats (Woodbury, 1974), but this explanation is not as probable since chlorpyrifos is highly lipophilic (Chambers and Carr, 1993) and could readily cross even adult blood–brain barriers. On the other hand, there are elimination mechanisms present in the blood, specifically, CaE and A-esterase, which become more active as the rat matures (described below). It is also possible that the ratio of

FIG. 5. Motor activity counts over a 30-min session, expressed as percentage of appropriate control values, plotted as a function of brain ChE inhibition, expressed as percentage of inhibition from appropriate control values. Peak effects are shown for each age group: 6.5 h for PND17 pups, 3.5 h for PND27, 3.5 h for adult males, and 6.5 h for adult females. Numbers beside each point indicate dose group (mg/kg).

DEVELOPMENT OF CHLORPYRIFOS SENSITIVITY

FIG. 6. Developmental profile of CaE (top) and CPFO-ase (bottom) activity in male rat liver and plasma. Data are expressed as percentage of adult male value as a function of age, and dashed line indicates 100% of the adult value (adults values were CaE, liver 96.1 6 15.0, plasma 0.61 6 0.05; CPFO-ase, liver 34.1 6 0.6, plasma 12.2 6 0.2, expressed as mean 6 SEM nmol/min/mg tissue or ml plasma). Insets show adult male and female control values for both enzymes (data from Chanda et al., 1997; Mortensen, 1997).

does not play a role in the age and gender differences documented in the present study. One likely factor in this age-related sensitivity is the difference that exists between young and adult animals in the detoxication rates of OP-oxons, which are mediated in part by CaE and A-esterase. CaEs tightly bind some OP-oxons, thus removing them from the pool available to affect AChE. The biological relevance of this mechanism is illustrated by studies wherein pretreatment with CaE inhibitors potentiates, and the administration of exogenous CaE attenuates, the effect of highly toxic OPs such as soman (Clement 1984; Maxwell, 1992a; Maxwell et al., 1987; Jimmerson et al., 1989). Studies from this laboratory have shown that chlorpyrifos inhibits CaE in vivo and that adding hepatic tissue (as a source of CaE) to

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brain tissue in vitro effectively lessens the ability of the chlorpyrifos oxon to inhibit AChE (Chanda et al., 1997). The relationship between A-esterase activity and OP-induced toxicity has been demonstrated by comparing species differences in sensitivity and by studies showing protection through treatment with exogenous A-esterase (Brealey et al., 1980; Costa et al., 1990; Li et al., 1993; reviewed in Costa and Manzo, 1995). To establish the influence of CaE and A-esterase on the inactivation of chlorpyrifos in developing rats, it is important to understand their developmental profiles. The ontogeny of CaE activity shown here generally agrees with others using different tissues and/or substrates (Atterberry et al., 1997; Mendoza, 1976; Morgan et al., 1994; Sterri et al., 1985), but the gender differences in CaE activity appear to depend somewhat on the substrate used (Sterri et al., 1985). In this study using p-nitrophenyl acetate, we report that females have less liver CaE activity than males; this agrees with Morgan et al. (1994) who used the same substrate in liver microsomes. While young animals have been shown to have lower levels of paraoxonase (Atterberry et al., 1996; Benke and Murphy, 1975) and CPFO-ase (Li et al., 1997; Mortensen et al., 1996) activity relative to adults, no profile of tissue CPFO-ase activity in developing animals has been published previously. PND27 rats were about twice as sensitive to chlorpyrifos as adults; at that time CPFO-ase had reached adult levels, yet PND21 rats still had only 40–50% of adult levels of CaE. Adult female rats, who were generally more sensitive than males, had half the liver CaE activity but higher plasma CPFO-ase activity. Thus, the age- and gender-related changes in sensitivity to chlorpyrifos correspond with the developmental pattern of esterase activity, especially that of CaE. We hypothesize that one mechanism for the greater sensitivity of younger rats to chlorpyrifos toxicity is their relative inability to detoxify chlorpyrifos through binding to CaE and hydrolysis by CPFO-ase. Given this marked age-related sensitivity to chlorpyrifos in rats, one obvious concern would be whether these data would predict age-related sensitivity to chlorpyrifos in humans. It has been shown that young children have less serum paraoxonase (i.e., A-esterase) activity than adults (Ecobichon and Stephens, 1973). Furthermore, A-esterase activity shows a genetic polymorphism in the adult population, with an 11-fold variation in paraoxonase activity (which displays a bimodal distribution) and 5-fold variability in CPFO-ase activity (Furlong et al., 1988). Children may have less serum CaE activity (Augustinsson and Barr, 1963; Ecobichon and Stephens, 1973), although the definition of CaE activity in those studies was not precise and may have been confounded by A-esterase activity. Taken together, these data seem to indicate that young humans may be more sensitive to chlorpyrifos toxicity than their adult counterparts for the same reason that we hypothesize young rats are more sensitive: the young appear to possess less detoxification esterases. Adult humans, however, have very little CaE activity compared to mice (Kaliste-Korhonen et al., 1996). One would therefore have to temper the above conclu-

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sion given the possibility that rodents may be a poor model for the human detoxification of chlorpyrifos. For example, assuming that most of the age-related sensitivity in rats is due to the developmental profile of CaE, and humans may have less CaE activity, it is possible that humans may be more sensitive to acute chlorpyrifos toxicity, but the age-related toxicity to acute doses may not be as exaggerated as in the rat. To confirm this, more information is needed from humans of all ages on the tissue levels as well as binding affinities of CaE or the Km values for A-esterase-mediated hydrolysis. The behavioral and biochemical information provided by this and other studies from our laboratory adds to the understanding of possible toxicokinetic mechanisms underlying the greater sensitivity of developing animals to chlorpyrifos toxicity. Other immature systems, such as cholinergic receptors, may also play a role in differential sensitivity. Our data, however, most strongly implicate the esterases (carboxylesterase, A-esterase) in determining age and gender differences in response to chlorpyrifos. This is essentially the same conclusion presented by Benke and Murphy (1975) using another OP, parathion. The kinetic mechanisms may apply only to ChE inhibitors that are highly detoxified by esterases, as is chlorpyrifos. In other words, the kinetic parameters of OPs may predict, to some extent, age-related differences in OP toxicity. ACKNOWLEDGMENTS The authors thank Deborah L. Hunter, Rene´e S. Marshall, Katherine L. McDaniel, and Pamela M. Phillips for their excellent technical help and Dr. Stanley Barone Jr. for supplying us with animals used for the developmental esterase study. Portions of this study have been presented at the 37th annual Society of Toxicology meeting, March 1998. This manuscript has been reviewed by the National Health and 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 does mention of trade names or commercial products constitute endorsement or recommendation for use.

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