Time-Course, Dose-Response, and Age Comparative Sensitivity of N-Methyl Carbamates in Rats

TOXICOLOGICAL SCIENCES 114(1), 113 123 (2010) doi:10.1093/toxsci/kfp286 Advance Access publication November 24, 2009 Time-Course, Dose-Response, and Age Comparative Sensitivity of N-Methyl Carbamates in Rats Virginia C. Moser,*,1 Katherine L. McDaniel,* Pamela M. Phillips,* and Anna B. Lowit *Toxicity Assessment Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711; and Health Effects Division, Office of Pesticide Programs, U.S. Environmental Protection Agency, Washington, District of Columbia 20460 1 To whom correspondence should be addressed at Toxicity Assessment Division (MD B105-04), 109 TW Alexander Dr., U.S. Environmental Protection Agency, Research Triangle Park, NC 27711. Fax: (919) 541-2922. E-mail: moser.ginger@epa.gov. Received October 9, 2009; accepted November 13, 2009 N-Methyl carbamate insecticides are reversible inhibitors of central and peripheral acetylcholinesterase (ChE). Despite their widespread use, there are few studies of neurotoxicity in young animals. To study potential age-related differences, we evaluated seven carbamates (carbaryl, carbofuran, formetanate, methiocarb, methomyl, oxamyl, and propoxur) in preweanling (17 days old or postnatal day [PND] 17) male rats. Motor activity was monitored, and ChE inhibition was measured in brain and red blood cells (RBCs) using a radiometric assay that minimized reactivation of ChE. First, we conducted time-course studies in PND17 Long- Evans male rats, using a single oral dose of each carbamate. Almost all carbamates showed maximal ChE inhibition at a 45-min time point; only methomyl showed an earlier peak effect (15 min). At 24 h, most inhibition had recovered. Next, doseresponse data were collected for each carbamate, using four doses and control, with motor activity testing beginning 15 min after dosing and tissue collection at 40 45 min. RBC ChE was generally inhibited to a greater degree than brain. Motor activity was not as sensitive a measure for some of the carbamates, with some differences across carbamates in the shapes of the dose-response curves. Additional studies documented age-related differences by comparing ChE inhibition in PND11, PND17, and adult rats following administration of carbaryl or carbofuran. Only the youngest (PND11) rats were more sensitive than adults to carbaryl, but both younger ages showed more effects than adults with carbofuran. Comparisons of the other carbamates to previous studies in adult rats suggest similar age-related sensitivity. Thus, these data show the time-course and dose-response characteristics for each carbamate and document greater sensitivity of the young for carbofuran and carbaryl. Key Words: neurotoxicity; N-methyl carbamates; acetylcholinesterase; motor activity; age sensitivity. Disclaimer: The information in this document has been reviewed by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents necessarily reflect the views of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use. The N-methyl carbamate pesticides inhibit acetylcholinesterase (ChE), producing cholinergic overstimulation, autonomic and neuromuscular dysfunction, and at high doses, result in coma and death. While they have been widely studied for decades, almost all such studies have used only adult laboratory animals, and there are few data concerning relative sensitivity in the young. While there is an extensive literature reporting age-related differences in toxicity in young rats exposed to organophosphorus (OP) pesticides, which also inhibit ChE (e.g., Brodeur and DuBois, 1963; Moser et al., 1998; Pope et al., 1991; Vidair, 2004), considerably less attention has been directed to similar characteristics of carbamates. Public concern regarding age-related sensitivity to the effects of pesticides led to the passage of the Food Quality Protection Act in 1996, which mandated greater assurance of protection for the young. Understanding potential age-related sensitivity is especially important in light of data suggesting greater exposure of children to pesticides (Boon et al., 2008; Fenske et al., 1990; Gamlin et al., 2007; Lu et al., 2000, 2006; National Research Council, 1993; Simcox et al., 1995). The most prominent difference between carbamates and OPs is the interaction with the ChE enzyme. The phosphorylated enzyme is reactivated very slowly, on the order of days to weeks. On the other hand, the carbamylated enzyme is much more labile and reactivates quickly, on the order of minutes to hours (Aldridge and Reiner, 1975). As a result, ChE activity recovers and metabolism clears most carbamates within 12 h (Dorough, 1970). This decarbamylation is increased by factors such as dilution and temperature, leading to issues with the assay used to measure cholinesterase activity. The use of a radiometric method (Johnson and Russell, 1975) or modified spectrophotometric method (Hunter et al., 1997; Nostrandt et al., 1993) improves the ability to assess ChE inhibition ex vivo following carbamate exposure. Unfortunately, very few studies of carbamates have taken this precaution. However, Ó The Author 2009. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

114 MOSER ET AL. such procedures are critical for understanding acute behavioral changes as a function of ChE inhibition, especially when such evaluations are made in the same subject. To evaluate better the common mode of action of N-methyl carbamates, we have initiated studies to systematically characterize and correlate their behavioral and biochemical effects in terms of time-course and dose-response. Studies of carbaryl, carbofuran, formetanate, methiocarb, methomyl, oxamyl, and propoxur in adult male rats have been published (McDaniel et al., 2007; Padilla et al., 2007). These studies have been extended to include preweanling rats, as previously used to evaluate age comparisons for OPs (Moser, 1999, 2000). The goals of the current studies were to (1) systematically compare time-course and dose-response characteristics of brain and red blood cell (RBC) ChE inhibition in postnatal day (PND) 17 rats, (2) compare these dose-response data of two of these carbamates in younger (PND11) and adult rats, and (3) determine behavioral effects as measured by locomotor activity in PND17 rats. METHODS Chemicals. Carbamates were obtained from Chem Service Inc. (West Chester, PA), all at purity 99%. Formetanate, oxamyl, and methomyl were dissolved in deionized water, whereas carbaryl, methiocarb, and propoxur were suspended in corn oil. Carbofuran was dissolved in a small amount of acetone (2.5% of final volume) and then suspended in corn oil. Control rats were administered the appropriate vehicles for each. All dosing solutions were prepared on the day of dosing. For the ChE assay, [ 3 H]acetylcholine (ACh) iodide (76 Ci/mmol; Perkin Elmer Life Sciences, Boston, MA) and other reagents (Sigma-Aldrich, St Louis, MO) were obtained at reagent-grade purity. Animals. Long-Evans hooded timed-pregnant or adult male rats were obtained from Charles River Laboratory (Raleigh, NC). They were housed on hardwood chip bedding (Beta-Chip) in the AAALAC International-accredited animal facility with regulated temperature (72 C ± 2 C) and humidity (50 ± 20%). Pregnant rats also had either a cotton pad (Nestlet) (time-course studies) or Enviro-Dri (dose-response studies) in each cage to serve as nesting material. Food (Purina Formulab Diet #5008 for dams and Purina Rodent Chow #5001 for adult males) and water (filtered tap) were freely available. Pregnant rats were allowed to deliver naturally; day of birth is considered PND0. On PND4 (time-course), PND3 (PND11 studies), or PND2 (doseresponse), all pups were grouped by sex and redistributed to the dams, assuring that littermates were spread across litters. All litters were culled to eight pups, with six males in each. Only males were used in these studies. Pups in each litter were dosed in a split-litter design, i.e., with no more than one pup within a litter receiving the same treatment. For the time-course studies, all six males in a litter were dosed, and for the dose-response studies, five males in a litter were dosed to assure that no more than one pup in a litter received the same dose. General observations of the litters indicated that dams did not treat the pups differently based on their dosing conditions. For the adult studies, male rats were received at 90 days of age and tested within 1 week. In-life testing. Body weight on the day of dosing was used to calculate dosing volume at 2 ml/kg. Within each litter, pups were randomly assigned to treatment groups. Adult rats were also weighed a few days after arrival and assigned to dose group using a stratification process by weight. All chemicals were administered via oral gavage at 2 ml/kg to provide greater accuracy in the dosing volume. Most human exposure to carbamates is expected to be via the diet or water consumption. TABLE 1 Doses (milligrams per kilogram) and Ages Tested for the Dose-Response Studies of Each Carbamate Name Ages Doses Carbaryl PND11, PND17, adult 0, 3, 7.5, 15, 30 Carbofuran PND11, PND17, adult 0, 0.1, 0.3, 0.6, 1 Formetanate PND17 0, 0.1, 0.3, 0.75, 1.5 Methiocarb PND17 0, 0.5, 2, 5, 10 Methomyl PND17 0, 0.25, 0.75, 1.25, 2.5 Oxamyl PND17 0, 0.07, 0.2, 0.5, 0.8 Propoxur PND17 0, 0.3, 1, 3, 10 Range-finding studies were conducted with each carbamate, in which one to three dose levels were administered to separate animals (two to four per dose) to determine doses which produce moderate but not extreme signs of toxicity. Doses for the subsequent studies were based on these results. Time-course and dose-response studies were conducted for each carbamate in PND17 rats; additional ages (adult, PND11) were included for carbaryl and carbofuran. Dosing was spaced so that sacrifice and tissue collection can take place at the same approximate time after dosing for all pups within the treatment group. To the extent possible, treatments were counterbalanced across the days of testing (note, the treatment groups for the longer time points were dosed earlier in the day). The time-course and dose-response studies for each carbamate were conducted separately. For the time-course, rats (n ¼ 6 per dose at each time) were dosed with either vehicle or a single dose of one of the carbamates (carbaryl 30 mg/kg, carbofuran 1 mg/kg, formetanate 3 mg/kg, methiocarb 12 mg/kg, methomyl 2.5 mg/kg, oxamyl 0.5 mg/kg, and propoxur 10 mg/kg). Nominal time points for the time-course study were 15, 45, 90, 180, or 1440 min (24 h); in practice, precise times were 15 20, 45 55, 90 95, 180 190, and 1440 1450 min after dosing. Vehicle-treated control rats were included only at 45, 180, and 1440 min. For the dose-response study, rats (n ¼ 10 per dose for PND17, n ¼ 8 per dose for PND11, and n ¼ 6 per dose for adult) were dosed with one of five doses of each carbamate as listed in Table 1. For PND17 rats, motor activity measurements were included in the dose-response study. Fifteen minutes after dosing, rats were placed in activity chambers shaped like a figure eight (Reiter, 1983). Photobeams spaced around the chamber detected movement as counts for a total of 20 min. Immediately after the activity session, rats were euthanized for tissue collection (40 45 min after dosing). For the PND11 and adult studies, rats remained in the home cage until euthanasia in the same time frame. Cholinesterase assay. At the appropriate time, rats were decapitated quickly under CO 2 -induced anesthesia. Trunk blood was collected in heparinized tubes. The whole brain was removed from the skull, split sagitally (including cerebellum), and placed in dry ice. Whole blood was spun at 1000 3 g for 10 min, and RBCs were collected and diluted with chilled 0.1M NaPO 4, ph 8.0/1.0% Triton buffer at a 1:2 dilution (one to two parts), and then placed in dry ice. Tissues were stored at 80 C until the day of assay. A radiometric assay was used to determine brain and blood ChE activity (Johnson and Russell, 1975). On the day of assay, brain tissues and RBC were thawed on ice (about 20 min). Brains were diluted in two volumes of chilled 0.1M sodium phosphate buffer (ph 8.0) with 1% Triton X-100 and homogenized for 20 30 s (Polytron homogenizer, Kinematica Model PT3100, Littau, Switzerland). RBC samples were used directly as prepared on the day of collection. The assay was conducted with a small reaction volume (20 ll sample plus 80 ll substrate) to minimize tissue dilution. The final ACh iodide concentration was 1.2mM. Reactions took place in a water bath at 26 C; incubation times were 30 s for brain and 2 min for RBC (lower ChE activity in RBC required longer times to produce reliably measured hydrolysis product).

NEUROTOXICITY OF CARBAMATES IN PND17 RATS 115 The reaction was then stopped using acid buffer. A toluene-based scintillant was added, the vials were shaken to allow extraction of the labeled [ 3 H] acetate, and [ 3 H] activity was counted within a few hours in a Beckman scintillation counter (model LS6000LL; Fullerton, CA). All samples were run in duplicate; duplicates > 20% apart were not used (this only occurred in 0.5% of the total samples assayed). Negative values after blank subtraction were set to zero. Statistical analyses. In the time-course studies, it was of interest to determine (1) significant differences between treated groups at each of the specific time points on the day of dosing and (2) persistent differences between the treated and control groups 24 h after dosing. To accomplish the first goal, data for carbamate-treated rats at each time point on the day of dosing were first subjected to two-way ANOVAs with tissue (brain and RBC) as one factor and time as a repeated (within-subject) measure. Since in all seven analyses the overall tissue-by-time interactions were highly significant (all ps < 0.0001), each tissue was then analyzed separately. Where the resultant overall F-test was significant (p < 0.05), it was followed by Tukey s Honestly Significant Difference (HSD) test to provide multiple comparisons across time points while maintaining alpha protection. For the second goal, the 24-h treated and control groups for each tissue were compared separately (simple t-test, total of 14). For all ChE analyses, data were analyzed using the actual values (i.e., not percent of control). Motor activity dose-response data were analyzed using a one-way ANOVA across dose groups. Dunnett s t-test was used as a protected (i.e., only after a significant overall F-test, p < 0.05) multiple comparison procedure to provide comparisons between the control and each dose group. To evaluate motor activity changes as a function of ChE inhibition (as was conducted in McDaniel et al., 2007), correlations were assessed by fitting a linear regression to the data and noting the r 2 value as a measure of goodness of fit (Supplementary figs. 1 7). Finally, to better assess comparability across studies, a post hoc analysis of the control motor activity data was conducted using a one-way ANOVA followed by Tukey s HSD test for pairwise comparisons across control groups. Dose-response data for ChE activity were analyzed using two approaches (1) traditional ANOVAs to evaluate significant dose-response differences between brain and RBC and to establish dose groups that were significantly different from control and (2) dose-response functions by fitting the data and determining point estimates for doses producing specific levels of ChE inhibition. As with the time-course data, initial two-way ANOVAs were conducted with tissue (brain and RBC) as a within-subject factor and dose as the second factor. Following significant (all ps < 0.0001) tissue-by-dose interactions for all 11 analyses, one-way ANOVAs for each tissue were then conducted with Dunnett s t-test to determine dose groups that were different from control. To obtain point estimates, data were fitted to a four-parameter logistic curve using Proast 17.04 (downloaded from http://www.rivm.nl/en /foodnutritionandwater/foodsafety/proast.jsp) (Edler et al., 2002; Slob, 2002). For estimation of the benchmark dose (BMD), the models are reparameterized so that the BMD is one of the model s parameters; this allows calculation of confidence intervals around that estimate. Point estimates for 10% (BMD10) and 50% (BMD50) inhibition of ChE were calculated with 95% confidence intervals (Supplementary figs. 8 52). RESULTS In the time-course studies, lethality occurred in one methiocarb-treated and one carbofuran-treated rat; none occurred in the dose-response studies. In general, high-dose pups occasionally displayed mild tremors; however, systematic observations were not possible since the animals were not in view while in the motor activity chambers, and they were euthanized as soon as they were removed. There was internal consistency across the studies in that the degree of ChE inhibition in the time-course studies was similar to the inhibition produced when the same dose was used in the dose-response studies. Time-Course ChE activity for the control groups in the time-course studies was very similar within each study as well as between studies (range, brain 4.98 5.803 lmol ACh hydrolyzed/min/mg tissue and RBC 0.678 0.896 lmol/min/ml) with the exception of the carbaryl study that had somewhat higher brain ChE activity (mean, 6.651 lmol/min/mg). There were no marked differences between control ChE activities on the day of dosing compared to the 24-h time point. For all carbamates, brain and RBC ChE showed considerable inhibition (brain, 30 70% inhibition and RBC, 60 90% inhibition) on the day of dosing, as shown in Figure 1. Treated groups were not significantly different from each other from 15 to 180 min for carbaryl-induced inhibition of brain and RBC ChE and for carbofuran inhibition of RBC. In contrast, methomyl showed the most rapid recovery during that time frame. The time of peak effect of each carbamate ranged from 15 to 90 min for both compartments. Based on statistically significant differences across the time points, the peak inhibitions were obtained at: carbaryl brain and RBC, 15 180 min; carbofuran brain, 15 45 min, and RBC, 15 180 min; formetanate brain, 45 90 min, and RBC, 15 90 min; methiocarb brain, 15 90 min, and RBC, 15 45 min; methomyl brain and RBC, 15 min; oxamyl brain and RBC, 45 90 min; and propoxur brain, 15 90 min, and RBC, 15 45 min. However, it should be noted that while the use of Tukey s test does provide alpha protection in multiple comparisons, small but significant differences at certain times may not be reproducible. Our choice of 40 45 min for the dose-response was based on these data as well as our need to have a common test time for all carbamates. Recovery of ChE activity to control levels was evident at 24 h for all except carbaryl RBC ChE (13% inhibition, p ¼ 0.0173) and methomyl brain ChE (6% inhibition, p ¼ 0.0385). In both cases, the degree of inhibition at 24 h was marginal. Dose-Response PND17 control ChE values obtained across the doseresponse studies were similar and compared well with those data from the time-course studies (range, brain 4.98 5.79 lmol ACh hydrolyzed/min/mg tissue and RBC 0.686 1.11 lmol/ min/ml). Brain ChE activity in controls was lowest in PND11 pups (3.38 3.70 lmol/min/mg tissue) and highest in adults (6.38 6.69 lmol/min/mg tissue), whereas there was little difference across ages for RBC ChE in untreated animals (PND11 0.635 0.871 and adults 0.606 0.599 lmol/min/ml). In contrast to the ChE data, there were clear study-to-study differences in control motor activity levels. Controls in the formetanate study were the highest, being significantly

116 MOSER ET AL. FIG. 1. Time-course of brain and RBC ChE inhibition produced by carbaryl, carbofuran, formetanate, methiocarb, methomyl, oxamyl, and propoxur in PND17 rats, expressed as % control (mean ± SEM). On the day of dosing, dose groups that are not significantly different (as indicated by Tukey s HSD multiple comparisons test) are indicated by the same letters. Asterisk indicates a significant difference between the control and treated group (t-test) at 24 h. For each carbamate, n ¼ 6 per dose per time point.

NEUROTOXICITY OF CARBAMATES IN PND17 RATS 117 FIG. 2. Dose-response of brain and RBC ChE inhibition produced by carbaryl or carbofuran in PND11, PND17, or adult rats, expressed as % control (mean ± SEM). Asterisk indicates dose groups significantly different from control (Dunnett s t-test). For both carbamates, PND11, n ¼ 8 per dose; PND17, n ¼ 10 per dose; adults, n ¼ 6 per dose. different than all the other control groups. The values of the carbofuran and methomyl controls were the lowest, but these differences were not statistically significant different from the other groups (except formetanate). All carbamates produced monotonic dose-related decreases in brain and RBC activity, as shown in Figures 2 and 3. BMDs producing 10 and 50% inhibition are listed in Table 2 for each age tested with carbaryl and carbofuran and in Table 3 for the carbamates tested only in PND17 rats. The carbaryl dose-response data (Fig. 2) showed a significant interaction across ages and tissue (dose-by-age-by-tissue F(8,103) ¼ 2.84, p ¼ 0.0068), triggering separate step-down analyses for each age. Brain ChE activity was significantly lower than control in all PND11 dose groups, whereas the lowest dose group was not different from control in the PND17 and adult rats. RBC ChE was inhibited in all dose groups in all three age groups. The greater sensitivity of the PND11 pups is reflected in the lower BMD values for brain inhibition in PND11 rats and very similar values for the adult and PND17 rats (Table 2). The BMD50 values for RBC inhibition are similar across ages, whereas the adult BMD10 value is lower than that of the younger rats. This perhaps reflects the lack of data at doses producing less inhibition, which would have improved the curve fitting. Motor activity (Fig. 4) was decreased only by the highest dose, 15 mg/kg. Motor activity depression was not well predicted by the regression model based on either brain or RBC ChE (brain r 2 ¼ 0.228 and RBC r 2 ¼ 0.203). The carbofuran ChE data showed a significant interaction of dose, age, and tissue (F(8,105) ¼ 4.05, p ¼ 0.0003) (Fig. 2). For brain ChE following carbofuran treatment, all dose groups at all ages were significantly lower than control activity, whereas the lowest dose was not different from control for adult RBC ChE. There was a clear separation of the age groups in the brain ChE dose-response, but the PND11 and PND17 data for RBC inhibition were similar. These age differences are reflected in the BMD10 and BMD50 values presented in Table 2. Since even the lowest dose produced considerable ChE inhibition (~50% in RBC for preweanling rats), the confidence limits for the BMD10 values span several orders of magnitude, reflecting considerable uncertainty in that estimate. In the motor activity data, the control group itself showed low

118 MOSER ET AL. FIG. 3. Dose-response of brain and RBC ChE inhibition produced by formetanate, methiocarb, methomyl, oxamyl, and propoxur in PND17 rats, expressed as % control (mean ± SEM). Asterisk indicates dose groups significantly different from control (Dunnett s t-test). For each carbamate, n ¼ 10 per dose. activity as compared to most of the other controls (Fig. 4). Regardless, all but the lowest dose significantly decreased motor activity below control levels. The monotonic doseresponse curve is reflected in the highest correlations between ChE inhibition and activity of all these carbamates, with brain r 2 ¼ 0.611 and RBC r 2 ¼ 0.626. As seen in Figure 3, formetanate decreased brain and RBC ChE in a treatment-related manner, and all doses were

NEUROTOXICITY OF CARBAMATES IN PND17 RATS 119 TABLE 2 Point Estimates for 10% ChE Inhibition (BMD10) and 50% Inhibition (BMD50) in PND17, PND11, and Adult Rats. Doses are milligrams per kilogram with 95% Confidence Limits in Parentheses Chemical Estimate Adult PND17 PND11 Carbaryl Brain BMD10 2.8 (1.2 4.9) 2.6 (0.93 4.9) 1.3 (0.46 2.6) BMD50 33.0 (22.9 80.2) 35.6 (25.9 61.0) 15.5 (11.6 21.3) RBC BMD10 0.25 (0.03 1.7) 0.87 (0.16 2.6) 0.99 (0.11 2.8) BMD50 6.8 (3.5 11.4) 10.7 (7.1 15.5) 8.3 (5.3 12.2) Carbofuran Brain BMD10 0.067 (0.033 0.12) 0.012 (0.0018 0.037) 0.0046 (0.00098 0.018) BMD50 1.0 (0.89 1.2) 0.37 (0.26 0.51) 0.19 (0.12 0.26) RBC BMD10 0.035 (0.0071 0.11) 0.0058 (0.00079 0.018) 0.0019 (0.00030 0.014) BMD50 0.37 (0.24 0.53) 0.098 (0.057 0.14) 0.084 (0.040 0.16) significantly different from control. The BMD10 and BMD50 values for RBC were somewhat lower than for brain, as presented in Table 3. For motor activity, the lowest and highest dose groups were significantly lower than control. However, as seen in Figure 4, the formetanate control group had much higher motor activity counts than the other control groups. A comparison across the controls and treated group averages for the other carbamates shows that the values for formetanatetreated groups fall within the no-effect levels, suggesting that the statistical significance observed may be a function of the high control group. In keeping with a lack of dose-response for motor activity, there was no predictability between motor activity and brain or RBC ChE inhibition (r 2 values < 0.01). Rats treated with the lowest dose of methiocarb (Fig. 3) showed similar inhibition (15 16%) of brain and RBC ChE, but only the brain was significantly different from control, reflecting the greater variability of the RBC measurement. At the higher doses, RBC and brain inhibition curves diverged, leading to similar BMD10 values for brain and RBC but lower BMD50 values for RBC (Table 3). The two higher doses significantly decreased motor activity (Fig. 4), although the magnitude of effect was similar in the two dose groups. Motor activity depression correlated somewhat with brain ChE inhibition (r 2 ¼ 0.489), whereas there was less predictability using the RBC ChE data (r 2 ¼ 0.357). The lowest dose of methomyl was not significantly different from control for either brain or RBC ChE (Fig. 3). The BMD10 and BMD50 values for RBC ChE inhibition were lower than those for brain, but the difference was greater with the BMD50 values. Motor activity (Fig. 4) was significantly increased over control, but only at the lowest dose, and higher doses were not different from control. However, the control values in this study were the lowest of the seven. Comparing the activity levels of the low dose group to the control and low dose groups TABLE 3 Point Estimates for 10% ChE Inhibition (BMD10) and 50% Inhibition (BMD50) in PND17 Rats. Doses are milligrams per kilogram with 95% Confidence Limits in Parentheses Chemical BMD10 BMD50 Formetanate Brain 0.058 (0.026 0.11) 0.46 (0.37 0.56) RBC 0.015 (0.0045 0.049) 0.28 (0.19 0.40) Methiocarb Brain 0.21 (0.036 0.67) 7.9 (5.4 86.9) RBC 0.18 (0.024 0.76) 2.8 (1.6 4.3) Methomyl Brain 0.40 (0.23 0.58) 3.1 (2.4 19.7) RBC 0.25 (0.086 0.47) 1.3 (1.1 1.6) Oxamyl Brain 0.075 (0.044 0.11) 0.51 (0.41 0.62) RBC 0.023 (0.0091 0.044) 0.18 (0.14 0.23) Propoxur Brain 0.55 (0.29 0.90) 5.9 (4.9 7.3) RBC 0.24 (0.076 0.76) 3.3 (2.4 4.5) FIG. 4. Dose-response of motor activity counts (mean ± SEM) during 20- min session produced by carbaryl, carbofuran, formetanate, methiocarb, methomyl, oxamyl, and propoxur in PND17 rats. Asterisk indicates dose groups significantly different from control (Dunnett s t-test). For each carbamate, n ¼ 10 per dose.

120 MOSER ET AL. with the other carbamates suggests that this increase is a consequence of the low control and not indicative of hyperactivity. The variability of motor activity as a function of brain or RBC ChE inhibition was moderate (brain r 2 ¼ 0.414 and RBC r 2 ¼ 0.357). For oxamyl, RBC ChE inhibition was significantly different from control in all dose groups, whereas the lowest dose did not produce significant inhibition of brain ChE (Fig. 3). Only the highest dose decreased motor activity levels (Fig. 4). There was very little concordance between motor activity depression and the degree of ChE inhibition (r 2 ¼ 0.112 and 0.059 for brain and RBC, respectively). All but the lowest dose of propoxur was significantly different from control for both brain and RBC ChE (Fig. 3). Only the highest dose (10 mg/kg) significantly decreased motor activity levels (Fig. 4). While the highest dose produced consistent motor activity depression along with ChE inhibition, there was more scatter in the motor activity counts in the lower dose groups. Goodness of fit was similar for motor activity versus brain ChE (r 2 ¼ 0.389) and versus RBC ChE (r 2 ¼ 0.335). DISCUSSION These studies reveal good concordance between brain and RBC ChE inhibition, with RBC being generally more sensitive, following a single dose of N-methyl carbamates in PND17 rats. Onset and peak effect were rapid, and recovery began within hours after a single dose. ChE was inhibited in a dosedependent manner, and for several carbamates, even the lowest dose tested produced significant inhibition. In contrast, motor activity dose-response curves were more variable and did not always show clear dose-related decreases. The youngest rats tested, PND11, were more sensitive as compared to adults to brain ChE inhibition produced by both carbaryl and carbofuran and to carbofuran-induced RBC ChE inhibition. The time-course of ChE inhibition was generally similar across most of the carbamates with the exceptions of methomyl, which showed significant recovery at the 45-min time point and almost complete recovery at 180 min, and oxamyl, which showed greater inhibition at 45 min compared to 15 min. The choice of 40 45 min for the dose-response data was optimal for all except methomyl, for which the test time may have been slightly later than the peak effect; however, this would only serve to underestimate the degree of toxicity. Decarbamylation of the ChE enzyme occurs at the same rate regardless of which carbamate is involved (Reiner, 1971), so differences in these time-course patterns are likely due to differing rates at which the chemical is removed from the target organ through metabolism and elimination. Generally, there was recovery at 24 h for all carbamates. The few instances of significant ChE inhibition at 24 h (carbaryl and methomyl) may have been type I errors due to the number of t-tests conducted or else due to the low variability of the concurrent control group since the magnitude of difference was marginal and within usual variability of controls. Our time-course data in PND17 rats agree well with the timecourse reported for the same carbamates by Padilla et al. (2007). In that study, adult male rats showed reversal of both brain and RBC ChE inhibition within hours of dosing, with full recovery at 24 h. Exact comparisons are not possible since the time points did not match exactly, but it generally appears that the PND17 rats recovered somewhat slower on the day of dosing. This was especially evident for carbaryl and formetanate (inhibition equal at 15 and 180 min in pups, recovery at 2 h in adults), oxamyl (peaked at 45 90 min in pups, recovery beginning at 30 min in adults), and propoxur (no difference between 15 and 90 min in pups, recovery beginning at 30 min in adults). In these studies, we were only able to conduct direct age comparisons for carbaryl and carbofuran. For both pesticides, the youngest age tested, PND11, was the most sensitive in terms of brain ChE inhibition for both carbamates and for RBC ChE inhibition for carbofuran. Interestingly, for carbofuran, the PND11 rats were more sensitive than PND17, which were more sensitive than adults, whereas the PND17 and adult rats were equally sensitive to carbaryl. Lethality produced by carbaryl was greater in weanling rats (23 days old) compared to adults (Brodeur and DuBois, 1963), but there was no difference in weanling (4 6 weeks old) and adult rats treated with methomyl (Gaines and Linder, 1986). A study comparing hypothermia in adult and PND17 rats treated with carbaryl reported that adults were actually more sensitive (Mack and Gordon, 2007). However, the PND17 rats, implanted with telemeters, were kept with the litter, which could have served to keep the pups warm and therefore could have attenuated the carbaryl-induced hypothermia. In summary, the present data are the first to report greater sensitivity of the young in terms of ChE inhibition produced by either carbaryl or carbofuran. Taking into account differences among the studies, we can learn more about age-related differences by comparing the present data in PND17 pups and those reported for adults (McDaniel et al., 2007; Padilla et al., 2007). In both sets of time-course studies, single doses of each carbamate were used, but these were not necessarily the same doses. However, similar doses of carbaryl (30 mg/kg) and methomyl (2.5 mg/kg in PND17 and 3 mg/kg in adult) were used, and the amount of inhibition produced was similar at both ages. Formetanate, methiocarb, and propoxur produced similar levels of ChE inhibition at both ages, yet the doses used were lower in the young rats compared to the adults (formetanate 3 vs. 10 mg/kg, methiocarb 12 vs. 25 mg/kg, and propoxur 10 vs. 20 mg/kg). For five of these carbamates (formetanate, methiocarb, methomyl, oxamyl, and propoxur), we can only compare our dose-response data to those collected in adult rats previously (McDaniel et al., 2007). The lowest dose of formetanate (0.1 mg/kg) did not decrease ChE activity in adults, yet produced significant inhibition in PND17 rats. Similarly, the magnitude of inhibition at higher doses was greater in the pups. There was

NEUROTOXICITY OF CARBAMATES IN PND17 RATS 121 also greater inhibition in pups compared to adults at similar doses for methiocarb, oxamyl, and propoxur but not methomyl. While these comparisons suggest age-related differences in sensitivity for at least some of the carbamates, such observations should be interpreted cautiously, as age-related differences in dose-response curves are best described using systematic comparisons in the same laboratory. Greater sensitivity of the young has been reported for another carbamate, aldicarb (Moser, 1999), and has been widely reported for several OP pesticides, including chlorpyrifos, parathion, malathion, and diazinon (Mendoza and Shields, 1977; Moser, 2000; Pope et al., 1991; Vidair, 2004). In terms of doses estimated to produce 50% brain ChE inhibition, aldicarb was about twofold more potent in PND17 pups compared to adults, which is comparable to the magnitude of difference reported herein between PND11 and adults treated with carbaryl. For carbofuran, the differences were greater, with the PND11 being about fivefold and PND17 threefold more sensitive than adults in terms of brain ChE inhibition. RBC BMD50s were about equal across ages for carbaryl but fourfold lower than adult values in both preweanling ages with carbofuran. Thus, age-related differences were found to be greater for carbofuran than for carbaryl and aldicarb. It has been suggested that this greater sensitivity of younger animals is probably due in part to lower detoxification ability in the young, particularly with respect to carboxylesterases and A-esterases. Carboxylesterases are known to play an important role in the elimination of carbaryl, carbofuran, aldicarb, and propoxur (Barata et al., 2004; Gupta and Dettbarn, 1993; Gupta and Kadel, 1989, 1990; McCracken et al., 1993), and carboxylesterase activity is lower in younger rats (Chanda et al., 1997; Moser et al., 1998); thus, greater toxicity could be expected in younger rats for these carbamates. There appear to be no reports in the literature describing the role of carboxylesterases in the detoxification of the other carbamates we have tested, nor could we find any information regarding A-esterase detoxification. While motor activity is a sensitive indicator of anticholinesterase toxicity in adult rats (McDaniel et al., 2007; Moser, 1995), this appears not to be the general case in preweanling rats. Visual inspection of the linear regressions fit to the ChE and activity levels (Supplementary figs. 8 14) used to derive the r 2 values did not reveal as good correlations as those seen in adults (McDaniel et al., 2007) nor did it reveal thresholds as was seen with chlorpyrifos (Nostrandt et al., 1997). Similar to the pattern seen here produced by methomyl, PND17 rats treated with aldicarb showed increased motor activity but no decreases when evaluated as percent of the corresponding control (Moser, 1999). Interestingly, in the reported aldicarb study, the control group showed lower activity levels (average, 84.2), but when the study was repeated, a higher control level was obtained (average, 111.3) and there were no activity changes significantly different from control. In addition, the young rats also showed fewer cholinergic signs of toxicity despite similar levels of ChE inhibition as compared to adults (Moser, 1999, 2000). Age comparisons with chlorpyrifos indicated that PND17 rats showed less motor activity depression at doses that produced much greater ChE inhibition than in adults (Moser, 2000). In contrast, dose-response curves overlapped for both motor activity and ChE inhibition in PND17 and adult rats following another OP, methamidophos (Moser, 1999). Thus, motor activity as a function of ChE inhibition is both age specific and chemical specific. These observations suggest that for PND17 motor activity studies, comparisons of treated rats to control should involve consideration of the actual control levels. Thus, additional research may be needed to determine more appropriate indicators of anticholinesterase toxicity in very young rats. Certain carbamates have been shown to act directly on cholinergic receptors as well as other neurotransmitter systems. For example, some carbamates interact directly with nicotinic receptors (Clarke et al., 1994; Nagata et al., 1997; Sherby et al., 1985; Smulders et al., 2003). The cholinergic nervous system function is not fully mature at 17 days (Ba and Seri, 1995), and some behavioral differences may reflect the relative influences and temporal development of these receptors. Furthermore, carbaryl alters catecholamine metabolism (Hassan, 1971; Ray and Poddar, 1986; Ray et al., 1984), as also might other carbamates. It is generally thought that OP cholinesterase inhibitory effects are modulated by other nonenzymatic actions to influence overall toxicity (Pope, 1999). Similar actions of carbamates could lead to some of the chemical-specific differences that have been described here. In summary, N-methyl carbamates produced ChE inhibition that peaked within minutes to hours after a single oral dose and recovered by 24 h. 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