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. Author manuscript; available in PMC: 2025 Sep 27.
Published in final edited form as: Toxicology. 2022 Sep 9;480:153317. doi: 10.1016/j.tox.2022.153317

The mechanistic basis for the toxicity difference between juvenile rats and mice following exposure to the agricultural insecticide chlorpyrifos

Katelyn N Sette 1, Navatha Alugubelly 1, Lauren B Glenn 1, Shirley X Guo-Ross 1, M Katherine Parkes 1, Juliet R Wilson 1, Caitlin N Seay 1, Russell L Carr 1,*
PMCID: PMC12465740  NIHMSID: NIHMS2107017  PMID: 36096317

Abstract

At high exposure levels, organophosphorus insecticides (OPs) exert their toxicity in mammals through the inhibition of brain acetylcholinesterase (AChE) leading to the accumulation of acetylcholine in cholinergic synapses and hyperactivity of the nervous system. Currently, there is a concern that low-level exposure to OPs induces negative impacts in developing children and the chemical most linked to these issues is chlorpyrifos (CPF). Our laboratory has observed that a difference in the susceptibility to repeated exposure to CPF exists between juvenile mice and rats with respect to the inhibition of brain AChE. The basis for this difference is unknown but differences in the levels of the detoxification mechanisms could play a role. To investigate this, 10-day old rat and mice pups were exposed daily for 7 days to either corn oil or a range of dosages of CPF via oral gavage. Four hours following the last administration of CPF on day 16, brain, blood, and liver were collected. The inhibition of brain AChE activity was higher in juvenile rats as compared to juvenile mice. The levels of activity of the detoxification enzymes and the impact of CPF exposure on their activity were determined in the two species at this age. In blood and liver, the enzyme paraoxonase-1 (PON1) hydrolyzes the active metabolite of CPF (CPF-oxon), and the enzymes carboxylesterase (CES) and cholinesterase (ChE) act as alternative binding sites for CPF-oxon removing it from circulation and providing protection. Both species had similar levels of PON1 activity in the liver and serum. Mice had higher ChE activity in liver and serum than rats but, following CPF exposure, the percentage inhibition was similar between species at an equivalent dosage. Even though rats had slightly higher liver CES activity than mice, the level of inhibition following exposure was higher in rats. In serum, juvenile mice had an 8-fold higher CES activity than rats, and exposure to a CPF dosage that almost eliminated CES activity in rats only resulted in 22% inhibition in mice suggesting that the high serum CES activity in mice as compared to rats is a key component in this species difference. In addition, there was a species difference in the sensitivity of CES to inhibition by CPF-oxon with rats having a lower IC50 in both liver and serum as compared to mice. This greater enzyme sensitivity suggests that saturation of CES would occur more rapidly in juvenile rats than in mice, resulting in more CPF reaching the brain to inhibit AChE in rats.

Keywords: Chlorpyrifos (CPF), Organophosphate insecticides, Developmental species differences

1. Introduction

One of the most common classes of agricultural pesticides is the organophosphorus insecticides (OPs). Earlier studies have reported a link between exposure to OPs and negative impacts on the brain of developing children (Eskenazi et al., 1999, 2007; Rauh et al., 2011, 2006, 2012). Preclinical studies using rodents have also reported impacts on behavior including deficits in memory, coordination, anxiety, and social behavior (Aldridge et al., 2005; Carr et al., 2020, 2017; Chanda and Pope, 1996; Johnson et al., 2009; Levin et al., 2010; Perez-Fernandez et al., 2020a, 2020b; Ricceri et al., 2003; Roegge et al., 2008; Timofeeva et al., 2008). The chemical most linked to these negative impacts in the literature is chlorpyrifos (CPF). After absorption into the body, CPF is metabolized to its active metabolite chlorpyrifos-oxon (CPF-oxon) by cytochrome P450. Following metabolism, CPF-oxon adversely affects the nervous system of animals and humans by inhibiting cholinesterase activity. At high exposure levels, CPF results in significant inhibition of neuronal acetylcholinesterase (EC 3.1.1.7, AChE) leading to an accumulation of acetylcholine (ACh) in the neuronal and neuromuscular synaptic junctions. This accumulation results in continual stimulation and hyperactivity of the nervous system.

Most environmental exposures to CPF occur at lower levels with no inhibition of AChE and thus, no hyperactivity of the nervous system suggesting the presence of a non-cholinergic target. With that in mind, our previous studies investigating the neurotoxic effects of developmental CPF exposure have utilized dosages of CPF that did not yield significant inhibition of brain AChE and utilized a repeated daily exposure paradigm to mimic environmental exposure in children. These studies utilized juvenile rats as the research model and we have observed that repeated exposure to these low dosages results in altered behavior (Carr et al., 2020, 2017). However, the basis for these changes is unclear.

To further this line of investigation, we had hopes of utilizing transgenic mouse models to identify the basis for the neurotoxic effects observed following low-level CPF exposure. Thus, we initiated a pilot dose-response study to determine the effects of repeated oral CPF exposure on brain AChE activity in wild-type juvenile mice. As presented in this study, we unexpectedly observed a significant species difference in the inhibition of brain AChE between juvenile mice and juvenile rats with greater AChE inhibition in juvenile rat brain than in juvenile mouse brain. This toxicity difference in juveniles differs from that observed in adults. Published literature has reported that adult rats (LD50 of 82–155 mg/kg) are more resistant to CPF toxicity than adult mice (LD50 of 60 mg/kg) (El-Sebae et al., 1978; Gaines, 1969; Garcia et al., 2006; Storm et al., 2000). Thus, the basis for the differences in the toxicity of CPF between juvenile rats and mice is not clear.

During exposure, CPF is converted to its active metabolite, CPF-oxon, which is not only a potent inhibitor of brain AChE but a potent inhibitor of other serine esterases such as the peripheral cholinesterases (ChE) and carboxylesterases (EC 3.1.1.1, CES) in non-target tissues. Peripheral ChE includes both the enzymes AChE and butyrylcholinesterase (EC 3.1.1.8; BChE) which are present in rodent and human liver and serum. The inhibition of these peripheral enzymes functions as a protective mechanism (Chambers et al., 1990; Clement, 1984; Maxwell, 1992) by stoichiometrically destroying a portion of the oxon thereby preventing it from reaching the brain to inhibit AChE. CPF-oxon can also be catalytically destroyed by paraoxonase 1 (PON1) which would also reduce the levels of circulating CPF-oxon. It is possible that a species difference exists in these detoxification mechanisms and contributes to the differences in brain AChE inhibition. Therefore, in this study, we measured the levels of these non-target esterases in the liver and serum of juvenile rats and mice and determined the impact that repeated oral CPF exposure has on ChE and CES activity levels.

2. Materials and methods

2.1. Chemicals

Chlorpyrifos (>99%) was a generous gift from DowElanco Chemical Company (currently known as Corteva, Inc., Indianapolis, IN). Chlorpyrifos-oxon and paraoxon (>97%) were synthesized as previously described (Chambers et al., 1990) and were generous gifts of the late Dr. Howard Chambers. All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).

2.2. Animals

Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) accredited, temperature-controlled environment (22 ± 2°C) with a 12-hour dark-light cycle with lights on between 0700 and 1900. Lab Diet rodent chow and tap water were freely available during the experiment. Adult male and female Sprague-Dawley CD rats and adult male and female C57BL/6 mice (Envigo, Indianapolis, IN) were used for breeding. Following parturition, male and female pups within the same litter were assigned to different treatment groups. Litters were not culled. During treatment, there was always a representative control animal present in each litter to match the CPF-treated animals. This control was of the same sex as the treated animals where possible. The day of birth was considered postnatal day 0 (PND0). All procedures were approved by the Mississippi State University Institutional Animal Care and Use Committee.

2.3. Animal treatment

CPF was administered daily by oral gavage to male and female rat and mice pups beginning on PND10 and continuing through PND16 (7 days). This age range is beyond the period of the growth spurt (PND7 and below) in rodents but encompasses a time of significant brain maturation and corresponds to the period following birth in humans (Andersen, 2003; Counotte et al., 2011; Tau and Peterson, 2010). CPF was dissolved in research-grade corn oil (Sigma, C8267, Lot #MKBS6944V) and delivered to the back of the throat using either a 10 μl (mice) or 25 μl (rats) tuberculin syringe equipped with a 1-inch 24-gauge straight intubation needle (Cadence Science, Cranston, RI). Mice pups received either corn oil or 1.0, 2.5, 5.0,or 10 mg/kg CPF, whereas rat pups received either corn oil or 0.5, 0.75, or 1.0 mg/kg CPF, both at a volume of 0.5 ml/kg. Each day, body weights were recorded and weight gain was calculated as the difference between the body weights on PND11–16 and the original body weight at the initiation of treatment on PND10. For rats, we utilized the brains collected in a previous study that had been treated with either corn oil, 2.5, or 5.0 mg/kg CPF using an identical treatment paradigm. These brains were used for the analysis of brain AChE activity. The body weight data had also been recorded and were included in the analysis for weight gain. Unfortunately, liver or blood were not collected from these rat pups and these endpoints were not available for use in this study. However, given that 1.0 mg/kg CPF greatly inhibits peripheral ChE and almost completely inhibits peripheral CES activity in rat pups, information on the effects of these dosages would not have added significant information. Using these samples allowed us to minimize animal use.

2.4. Enzyme assays

Mice and rats were sacrificed 4 h following the final administration of CPF on PND16 and the forebrain (whole brain minus medulla, pons, and cerebellum), serum, and liver were collected, immediately frozen on dry ice, and stored at − 80°C until assay. Brain and liver were homogenized in assay buffer (0.05 M Tris-HCl buffer (pH 7.4)) in a glass mortar using a Wheaton motorized grinder and a Teflon pestle.

With respect to litter distribution in mice, a total of 11 litters were used (Supplementary Table 1). Not all liver and serum enzyme activities were determined for all mice in these litters. With respect to litter distribution in rats, a total of 12 litters were used for brain AChE and 7–8 litters were used for the liver and serum enzymes (Supplementary Table 2). Supplementary Tables 1 and 2 contain the total number of pups in each litter, the number of mice/rats from each litter used for the determination of each parameter, and a further breakdown of the males and females from each litter used for the determination of each parameter. For the rat litters, not all tissue samples collected from rats in each litter were used for this project. Tissue samples from those littermates were used for other ongoing projects and, as necessary, the samples from one of two control pups in the litter were used in that project and were not included in this project. For the entire project, the final numbers of total replications and replications per sex for each treatment group for each enzyme for mice and rats can be found in Supplementary Table 3. A further breakdown of how many mouse and rat pups were used for each treatment group from each litter is presented for brain AChE, Liver ChE and CES, and serum ChE and CES (Supplementary Tables 4–8).

For determination of forebrain AChE and liver ChE activities, homogenates were diluted in the assay buffer to final concentrations of 1.0 and 0.25 mg/ml, respectively. Activities were measured using a discontinuous spectrophotometric assay using a modification (Chambers et al., 1988) of Ellman et al. (Ellman et al., 1961) using acetylthiocholine as the substrate (1 mM final concentration) and 5,5′-dithiobis (nitrobenzoic acid) as the chromogen. For determination of serum ChE activity, serum was diluted in assay buffer to a final concentration of 7.69 μl/ml. Activity was measured using a continuous spectrophotometric assay modified to a 96-well microplate as described by Ellman et al. (Ellman et al., 1961) using acetylthiocholine as the substrate (1 mM final concentration) and 5,5′-dithiobis (nitrobenzoic acid) as the chromogen. For all ChE assays, additional tubes/wells containing eserine sulfate (10 μM final concentration) were included in the assay to correct for non-enzymatic hydrolysis for each sample.

For determination of liver and serum CES activities, liver homogenates and serum were diluted in the assay buffer to working concentrations of 1.0 mg/ml and 6.66 μl/ml, respectively. Activity was measured using a spectrophotometric assay based on a previously described method (Carr and Chambers, 1991) that was modified to a continuous 96-well microplate assay. Briefly, 150 μl of the tissue (working concentration) was preincubated at 37°C for 5 min prior to the addition of prewarmed 150 μl substrate 4-nitrophenol valerate (to obtain a 0.1 mM final concentration). Continuing to incubate at 37°C, the absorbance at 405 nm was read every 20 s for 5 min and the slope was calculated. For each sample, additional wells containing paraoxon (10 μM final concentration) were included in the preincubation part of the assay to correct for non-enzymatic hydrolysis.

2.5. PON1-mediated hydrolysis of CPF

For determination of hydrolysis of CPF-oxon by PON1, liver and serum from experimentally naïve PND16 male and female rats and mice were used. For both species, there were 6–7 pups for each sex that were selected from 4 litters with no more the two of the same sex originating from the same litter. Livers were homogenized in assay buffer and homogenates and serum were diluted in either the assay buffer or the assay buffer + 1 mM EDTA to final concentrations of 1.0 and 0.5 mg/ml, respectively. PON1-mediated hydrolysis of CPF-oxon was determined by measuring the production of 3,5,6-trichloro-2-pyridinol based on a previously described method (Furlong et al., 1989) but adapted to a continuous assay on a microplate. Briefly, 200 μl of the tissue dilution (+/− EDTA) was preincubated at 37°C for 5 min prior to the addition of the substrate CPF-oxon (0.35 mM final concentration). Continuing to incubate at 37°C, the absorbance at 315 nm was read every 20 s for 5 min and the slope was calculated. For each sample, the wells containing EDTA were included to correct for non-PON1 hydrolysis.

For all tissues, protein concentrations were determined using the BCA reagent (ThermoPierce) with bovine serum albumin standards. Using either the absorbance units (discontinuous assay) or slopes (continuous assays), the specific activity of each enzyme from each sex of each species was determined as nmoles min−1 mg protein−1.

2.6. Carboxylesterase IC50 determination

IC50 values assays were determined based on previously published methods (Atterberry et al., 1997; Meek et al., 2021) but were adapted to a 96-well microplate. Tissue concentrations were determined that would yield similar control slope levels. Liver and serum from experimentally naïve PND16 male and female rats and mice (3 for each sex from 3 different litters) were used. Livers were homogenized in assay buffer and centrifuged to obtain the supernatant. It was diluted in assay buffer to obtain final concentrations of 1.0 and 0.5 mg/ml, respectively. Serum was diluted in assay buffer to obtain final concentrations of 0.25 and 0.025 μl/ml, respectively. Increasing concentrations of either CPF-oxon (5 μl) or the ethanol vehicle were pipetted into different wells of a 96-well plate. Diluted prewarmed tissue (150 μl) was added and incubated at 37°C for 30 min on a shaker. This was followed by the addition of 150 μl of the prewarmed substrate 4-nitrophenol valerate as described above. Absorbance at 405 nm was read every 20 s for 5 min and the slope was calculated. For each replication, additional wells containing paraoxon (10 μM final concentration) were included in the preincubation part of the assay to correct for non-enzymatic hydrolysis. IC50 values were calculated as described in Meek et al. (Meek et al., 2021).

2.7. Statistical analysis

Statistical analysis was performed using SAS statistical package (SAS Institute Inc., Cary, NC). The sphericity of the body weight gain data was initially tested by analysis of variance (ANOVA) using the general linear model with a repeated measures paradigm and was not found to violate the assumption of sphericity. Using a repeat measures paradigm, analysis of body weight gain was conducted by ANOVA using the Mixed procedure (Littell et al., 1996). The analysis identified significant differences in the main effects (sex, treatment, and day) and all possible interactions. Enzyme activities and IC50 values were also analyzed by ANOVA using the Mixed procedure. All analyses determined significant differences in sex, treatment, and sex × treatment interactions. Prior to analysis, the distribution of the conditional residuals for all data was evaluated for each parameter to determine the appropriateness of the statistical model. Any parameter not found to be sufficiently normally distributed was subjected to Blom’s transformation in SAS using the PROC RANK procedure with the NORMAL option (Altman, 1990; Blom, 1958). All analyses included litter and sex × treatment × litter as random effects. No sex differences were detected so males and females were pooled for final analysis. Mean separation was performed by least-square means. The criterion for significance was set at p < 0.05.

3. Results and discussion

No signs of cholinergic hyperactivity such as lacrimation, diarrhea, or tremors were observed at any time during CPF exposure in either species. In both species, there was a significant overall effect of day (p < 0.0001) indicating significant growth over the treatment period. There were no significant interactions. In rats, there was a significant effect of treatment on weight gain (p = 0.0214) but only the 5.0 mg/kg CPF treatment group was significantly different from controls (p = 0.0050). Lower level analysis indicated that these effects occurred on days 12–16 (Fig. 1A). This pattern of effect on weight gain is similar to that reported in our earlier studies (Carr et al., 2017, 2011, 2014). In contrast, there was no effect on weight gain in any of the treatment groups in mice even though a dosage of 10 mg/kg CPF was administered (Fig. 1B).

Fig. 1.

Fig. 1.

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Rates of Weight Gain in Juvenile Rats and Mice. Rat (A) and mouse (B) pups were exposed daily from postnatal day 10 through 16 to either corn oil (control) or various dosages of CPF. Values are expressed as weight ± SE (n = 8–16). Values significantly different from their respective control (p < 0.05) are indicated with an asterisk (*).

Based on our results, the control activities of AChE in juvenile rat and mouse brains are similar (Table 1). This finding agrees with adult studies comparing rat and mice brain AChE (Johnson and Wallace, 1987). In addition, the in vitro sensitivities of brain AChE to inhibition by OPs, such as paraoxon and chlorpyrifos-oxon, have been reported to also be similar in in vitro adult studies that studied both species (Coban et al., 2016; Johnson and Wallace, 1987). However, we observed that repeated daily oral exposure to CPF induced different patterns in the inhibition of brain AChE between juvenile rats and juvenile mice (Fig. 2). The lowest dosage of CPF that resulted in significant brain AChE inhibition in juvenile rats was 1.0 mg/kg which yielded 22% inhibition, whereas, this dosage did not result in any inhibition of brain AChE in juvenile mice. The highest dosage of CPF administered to juvenile rats was 5.0 mg/kg which yielded 63% inhibition of brain AChE inhibition. This dosage also yielded significant inhibition of brain AChE in juvenile mice but only at a level of 14%. A higher dosage of CPF (10 mg/kg) was administered in juvenile mice and yielded 50% inhibition. These data indicate that repeated daily oral CPF exposure results in greater brain AChE inhibition in juvenile rats than in juvenile mice.

Table 1.

Specific Activities of Enzymes from Juvenile Mouse and Rat.

Mouse Rat
Brain AChE 88.17 ± 2.58 89.07 ± 2.28
Liver ChE 153.02 ± 8.61 69.74 ± 3.41 *
Serum ChE 5960.94 ± 683.48 2779.10 ± 198.34 *
Liver CES 19,249.98 ± 745.89 37,804.30 ± 674.55 *
Serum CES 20,017.29 ± 757.44 2539.30 ± 95.31 *
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Data (nmoles min−1 mg protein−1) presented as mean ± SEM (n = 10–16).

*

= Significantly difference between species in row

Fig. 2.

Fig. 2.

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Brain Acetylcholinesterase. Percent of control of acetylcholinesterase specific activity in the brain of rat and mice pups exposed daily from postnatal day 10 through 16 to either corn oil (control) or various dosages of CPF. Values are expressed as mean ± SE (n = 8–16). Percent inhibition for each treatment group as compared to its respective control is presented in the oval overlaying the corresponding bar. Control values can be found in Table 1. Values significantly different from their respective control (p < 0.05) are indicated with an asterisk (*).

The first non-target esterase investigated was the effect of exposure on the activities of liver and serum ChE where repeated daily oral exposure to CPF resulted in a dose-response type pattern in both tissues (Fig. 3 A and 3B). For both liver and serum ChE, control juvenile mice possessed over 2-fold higher ChE activities than did control juvenile rats (Table 1). These differences in activity levels could have certainly contributed to the increased resistance in juvenile mice. However, at the same exposure level (1.0 mg/kg CPF), the inhibition of liver ChE in juvenile rat liver was 64%, while in juvenile mice, it was only 52% (Fig. 3 A). As with liver ChE, there was a similar percentage inhibition of serum ChE between rats (53%) and mice (41%) at the same level of exposure (1.0 mg/kg) (Fig. 3B). Overall, this suggests that the higher level of liver and serum ChE present in juvenile mice did not offer the juvenile mice a significantly greater level of protection as compared to juvenile rats.

Fig. 3.

Fig. 3.

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Liver and Serum Cholinesterase (ChE) Inhibition. Percent of control of cholinesterase specific activity in the liver (A) and serum (B) of rat and mice pups exposed daily from postnatal day 10 through 16 to either corn oil (control) or various dosages of CPF. Values are expressed as mean ± SE (n = 9–15). Percent inhibition for each treatment group as compared to its respective control is presented in the oval overlaying the corresponding bar. Control values can be found in Table 1. Values significantly different from their respective control (p < 0.05) are indicated with an asterisk (*).

The next non-target esterase investigated was the effect of exposure on the activity of liver CES where repeated daily oral exposure to CPF resulted in a dose-response type pattern (Fig. 4 A). Interestingly, for liver CES, control juvenile rats had almost a 2-fold higher CES liver activity than did control juvenile mice (Table 1). In addition, the IC50 value for rat liver CES was 3.6-fold lower than the same value for mouse liver CES indicating greater sensitivity of rat liver CES (Table 2). Based strictly on activity levels and enzyme sensitivity, if liver CES was playing the main protective role, juvenile rat liver should be more resistant to CPF exposure because it has twice the amount of enzyme activity. However, even with the higher activity, CES activity in juvenile rat liver was almost maximally inhibited (96%) at 1.0 mg/kg CPF, whereas, CES activity in juvenile mouse liver resulted in only 54% inhibition when exposed to the same dosage of CPF. This maximal inhibition in rats suggests that while liver CES does effectively remove CPF-oxon from circulation, it does not appear to be the key protective enzyme.

Fig. 4.

Fig. 4.

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Liver and Serum Carboxylesterase (CES) Inhibition. Percent of control carboxylesterase specific activity in the liver (A) and serum (B) of rat and mice pups exposed daily from postnatal day 10 through 16 to either corn oil (control) or various dosages of CPF. Values are expressed as mean ± SE (n = 9–15). Percent inhibition for each treatment group as compared to its respective control is presented in the oval overlaying the corresponding bar. Control values can be found in Table 1. Values significantly different from their respective control (p < 0.05) are indicated with an asterisk (*).

Table 2.

Carboxylesterase IC50s in Liver and Serum of Juvenile Mouse and Rat.

Mouse Rat
Liver   3.36 ± 0.16 0.93 ± 0.05 *
Serum 10.75 ± 0.19 8.79 ± 0.32 *
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Data (nM CPF-oxon) presented as mean ± SEM (n = 6).

*

= Significantly different from other species in row

The next non-target esterase investigated was the effect of exposure on the activity of serum CES where, as observed with the other enzymes, repeated daily oral exposure to CPF resulted in a dose-response type pattern (Fig. 4B). Control juvenile mice had almost an 8-fold higher serum CES activity than did control juvenile mice (Table 1). At the same exposure level (1.0 mg/kg CPF), serum CES activity in juvenile rats was almost maximally inhibited (95%) suggesting that the protective mechanism provided by the serum CES was saturated. However, the same dosage in juvenile mice resulted in only 22% inhibition suggesting that a significant level of enzymes remained active in the serum. It required raising the CPF dosage to 10 mg/kg in juvenile mice to approach saturation of the protective mechanism provided by serum CES. As evidenced by a significantly lower IC50 value, rat serum CES was statistically more sensitive to inhibition by CPF-oxon than was mouse serum CES (Table 2). However, the differences in the IC50s between rats and mice were not very large suggesting that this sensitivity difference probably did not play an important role (Table 2).

In addition to the esterases, another potential protective enzyme that could have played a role in the species difference is paraoxonase 1 (PON1) which catalytically hydrolyzes organophosphates such as CPF-oxon. However, the activity of PON1 in both liver and serum was similar between species suggesting that it did not play a role in the differences in sensitivity to CPF exposure (Table 3).

Table 3.

Chlorpyrifos-oxon Hydrolysis by PON1 in Liver and Serum of Juvenile Mouse and Rat.

Mouse Rat
Liver 627.62 ± 36.35 626.31 ± 19.87
Serum 811.02 ± 46.91 814.45 ± 28.73
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Data (nmoles min−1 mg protein−1) presented as mean ± SEM (n = 12–14).

*

= Significantly different from other species in row

Of the protective enzymes investigated, it appears that the differences in serum CES activity may play the most significant role in the toxicity differences between juvenile rats and mice during CPF exposure. Following oral exposure, a toxicant is absorbed by the intestine and enters the bloodstream via the hepatic portal vein which takes it to the liver. Normally, the liver is considered the main point of metabolism. However, Poet et al. (2003) reported that metabolism of CPF occurs in the intestine and, at low-dose oral exposures (such as those used in this study), that metabolism would contribute significantly to the overall activation of CPF. If a large amount of the CPF is converted to CPF-oxon in the intestine, the release of CPF-oxon into the circulation would initially occur in the hepatic portal vein where it would have the first opportunity to be bound by CES. This would allow the elimination of any CPF-oxon produced by the intestine before delivery to the liver. This would be beneficial to the species with a higher level of serum CES.

Hypothetically, let’s consider what occurred in rats and mice exposed to the same dosage of CPF (1.0 mg/kg). In rats, the CPF-oxon entering the hepatic portal vein saturated the serum CES thereby allowing CPF/CPF-oxon to reach the liver. Once in the liver, the CPF-oxon inhibited CES at that location, the CPF was converted to CPF-oxon by cytochrome P450, and together these saturated the liver CES. This allows more CPF/CPF-oxon to enter the general circulation and reach the brain. In mice, most of the CPF-oxon produced in the intestine is bound by serum CES such that mostly CPF reaches the liver. The CPF reaching the liver is converted to CPF-oxon but that amount is not sufficient to saturate the liver CES thus lower levels of CPF-oxon escape into the general circulation and are able to reach the brain.

The basis for the difference between juvenile rats and mice is still unclear. It is well known that adult rodents have higher levels of CES in their blood which is an important factor in why juveniles are more susceptible than adults to OP compounds such as CPF (Atterberry et al., 1997; Moser et al., 1998). It has also been reported that adult mice have 1.8-fold more CES activity in their blood than do adult rats (Rudakova et al., 2011). It is possible that mice may simply develop faster than rats and reach adult levels of blood CES activity much earlier than do rats. However, other factors may play a role in the different susceptibility of juvenile mice and rats to CPF exposure. These include species differences in the cytochrome P450-mediated activation and detoxification of CPF in the liver and/or intestine and differences in the intestinal PON1-mediated detoxication of CPF-oxon.

The current study utilized a repeated daily exposure paradigm that encompassed 7 days, which has been the main paradigm utilized in our laboratory. It is quite possible that this repeated exposure could have differentially altered the compensatory mechanisms between rats and mice with respect to how CPF is handled metabolically. For example, in adult rats exposed repeatedly to CPF for 5 days, the total hepatic microsomal cytochrome P450 content was significantly reduced by the exposure (Abdou et al., 2020). In mice repeatedly exposed to CPF for 5 days, there was a decrease in the capacity to metabolize testosterone to androstenedione or 2αOH-testosterone suggesting alterations in metabolism mediated by CYP2C11. However, there was no effect on the production of 6 other metabolites, and no effect on the metabolism of CPF to CPF-oxon was observed (Cometa et al., 2007). Hence, metabolism in adult animals is modified by repeated exposure to CPF and it is possible that the repeated exposure paradigm in juveniles could have produced species dependent changes in metabolism as well. If so, these types of things could have contributed to the species differences observed in our study. This also suggests that an acute exposure may have resulted in a different outcome with respect to species differences. An acute exposure would also not have been affected by any compensatory changes induced by the repeated exposure. However, the majority of childhood OP environmental exposures involve low-level chronic exposures and these are the exposure scenarios that have been repeatedly linked to negative health effects in children including neurological disorders (Moore et al., 2022; Roberts et al., 2019; Ross et al., 2013; Tessari et al., 2022). Based on the findings of the human epidemiological studies, our repeated exposure paradigm was designed to mimic this sort of “real world” exposure situation. Many other laboratories investigating the developmental effects of OP insecticides also utilize repeated exposure paradigms. In this study, we report that juvenile mice and juvenile rats are different with rats being more sensitive to repeated low-level oral CPF exposure. While the sensitivity endpoint was based on brain AChE inhibition, brain AChE is the canonical target for OP insecticides and its inhibition is a very good indicator that significant levels of CPF/CPF-oxon have been delivered to the brain through the circulation. Based on the differences in brain AChE inhibition we observed between rats and mice exposed to the same dosages, it can be assumed that greater amounts of CPF/CPF-oxon reached the juvenile rat brain than reached the juvenile mouse brain. If more CPF/CPF-oxon reaches the juvenile rat brain than the juvenile mouse brain, it is logical to assume that more damage will occur in the juvenile rat brain. In assessing the human health risk from pesticides, an important consideration during the dose-response assessment step is either identifying the highest exposure level at which harmful effects are no longer observed in test animals which is termed the No-Observed-Adverse-Effect Level (NOAEL) or identifying the lowest exposure level at which harmful effects are observed in test animals termed the Lowest-Observed-Adverse-Effect Level (LOAEL). Since we observed effects in the juvenile rat brain but not in the juvenile mouse brain at the same exposure level, one could hypothesize that the NOAEL and LOAEL will be lower in juvenile rats than in juvenile mice exposed repeatedly to low dosages of CPF.

Supplementary Material

Sette Supplement

Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.tox.2022.153317.

Acknowledgments

This research was supported by the Mississippi Agricultural and Forestry Experiment Station (MAFES), the College of Veterinary Medicine, Mississippi State University, and NIH 1R15ES023162 and NIH 1R15ES032959.

Footnotes

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRediT authorship contribution statement

Katelyn N. Sette: Investigation, Writing – original draft. Navatha Alugubelly: Investigation, Writing – review & editing. Lauren B. Glenn: Investigation, Writing – review & editing. Shirley X. Guo-Ross: Project administration, Writing – review & editing. M. Katherine Parkes: Investigation, Writing – review & editing. Juliet R. Wilson: Investigation, Writing – review & editing. Caitlin N. Seay: Investigation, Writing – review & editing. Russell L. Carr: Supervision, Conceptualization, Formal analysis, Visualization, Writing – original draft.

Data availability

Data will be made available on request.

References

  1. Abdou RH, Elbadawy M, Khalil WF, Usui T, Sasaki K, Shimoda M, 2020. Effects of several organophosphates on hepatic cytochrome P450 activities in rats. J. Vet. Med. Sci. 82, 598–606. 10.1292/jvms.19-0452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aldridge JE, Levin ED, Seidler FJ, Slotkin TA, 2005. Developmental exposure of rats to chlorpyrifos leads to behavioral alterations in adulthood, involving serotonergic mechanisms and resembling animal models of depression. Environ. Health Perspect. 113, 527–531. 10.1289/ehp.7867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Altman DG, 1990. Practical Statistics for Medical Research. Chapman & Hall/CRC, London. 10.1201/9780429258589. [DOI] [Google Scholar]
  4. Andersen SL, 2003. Trajectories of brain development: point of vulnerability or window of opportunity. Neurosci. Biobehav. Rev. 27, 3–18. 10.1016/s0149-7634(03)00005-8. [DOI] [PubMed] [Google Scholar]
  5. Atterberry TT, Burnett WT, Chambers JE, 1997. Age-related differences in parathion and chlorpyrifos toxicity in male rats: target and nontarget esterase sensitivity and cytochrome P450-mediated metabolism. Toxicol. Appl. Pharmacol. 147, 411–418. 10.1006/taap.1997.8303. [DOI] [PubMed] [Google Scholar]
  6. Blom G, 1958. Statistical Estimates and Transformed Beta Variables. John Wiley and Sons, Inc, New York, NY. 10.1002/bimj.19610030410. [DOI] [Google Scholar]
  7. Carr RL, Alugubelly N, de Leon K, Loyant L, Mohammed AN, Patterson ME, Ross MK, Rowbotham NE, 2020. Inhibition of fatty acid amide hydrolase by chlorpyrifos in juvenile rats results in altered exploratory and social behavior as adolescents. Neurotoxicology 77, 127–136. 10.1016/j.neuro.2020.01.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Carr RL, Armstrong NH, Buchanan AT, Eells JB, Mohammed AN, Ross MK, Nail CA, 2017. Decreased anxiety in juvenile rats following exposure to low levels of chlorpyrifos during development. Neurotoxicology 59, 183–190. 10.1016/j.neuro.2015.11.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Carr RL, Borazjani A, Ross MK, 2011. Effect of developmental chlorpyrifos exposure, on endocannabinoid metabolizing enzymes, in the brain of juvenile rats. Toxicol. Sci. 122, 112–120. 10.1093/toxsci/kfr081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Carr RL, Chambers JE, 1991. Acute effects of the organophosphate paraoxon on schedule-controlled behavior and esterase activity in rats: dose-response relationships. Pharmacol. Biochem. Behav. 40, 929–936. 10.1016/0091-3057(91)90108-e. [DOI] [PubMed] [Google Scholar]
  11. Carr RL, Graves CA, Mangum LC, Nail CA, Ross MK, 2014. Low level chlorpyrifos exposure increases anandamide accumulation in juvenile rat brain in the absence of brain cholinesterase inhibition. Neurotoxicology 43, 82–89. 10.1016/j.neuro.2013.12.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chambers H, Brown B, Chambers JE, 1990. Noncatalytic detoxication of six organophosphorus compounds by rat liver homogenates. Pestic. Biochem. Physiol. 36, 308–315. 10.1016/0048-3575(90)90040-9. [DOI] [Google Scholar]
  13. Chambers JE, Wiygul SM, Harkness JE, Chambers HW, 1988. Effects of acute paraoxon and atropine exposures on retention of shuttle avoidance behavior in rats. Neurosci. Res. Commun. 3, 85–92. [Google Scholar]
  14. Chanda SM, Pope CN, 1996. Neurochemical and neurobehavioral effects of repeated gestational exposure to chlorpyrifos in maternal and developing rats. Pharmacol. Biochem. Behav. 53, 771–776. 10.1016/0091-3057(95)02105-1. [DOI] [PubMed] [Google Scholar]
  15. Clement JG, 1984. Role of aliesterase in organophosphate poisoning. Fundam. Appl. Toxicol. 4, S96–S105. 10.1016/0272-0590(84)90141-6. [DOI] [PubMed] [Google Scholar]
  16. Coban A, Carr RL, Chambers HW, Willeford KO, Chambers JE, 2016. Comparison of inhibition kinetics of several organophosphates, including some nerve agent surrogates, using human erythrocyte and rat and mouse brain acetylcholinesterase. Toxicol. Lett. 248, 39–45. 10.1016/j.toxlet.2016.03.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Cometa MF, Buratti FM, Fortuna S, Lorenzini P, Volpe MT, Parisi L, Testai E, Meneguz A, 2007. Cholinesterase inhibition and alterations of hepatic metabolism by oral acute and repeated chlorpyrifos administration to mice. Toxicology 234, 90–102. 10.1016/j.tox.2007.02.008. [DOI] [PubMed] [Google Scholar]
  18. Counotte DS, Smit AB, Pattij T, Spijker S, 2011. Development of the motivational system during adolescence, and its sensitivity to disruption by nicotine. Dev. Cogn. Neurosci. 1, 430–443. 10.1016/j.dcn.2011.05.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. El-Sebae AH, Ahmed NS, Soliman SA, 1978. Effect of pre-exposure on acute toxicity of organophosphorus insecticides to white mice. J. Environ. Sci. Health B 13, 11–24. 10.1080/03601237809372074. [DOI] [PubMed] [Google Scholar]
  20. Ellman GL, Courtney KD, Andres V Jr., Feather-Stone RM, 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95. 10.1016/0006-2952(61)90145-9. [DOI] [PubMed] [Google Scholar]
  21. Eskenazi B, Bradman A, Castorina R, 1999. Exposures of children to organophosphate pesticides and their potential adverse health effects. Environ. Health Perspect. 107, 409–419. 10.1289/ehp.99107s3409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Eskenazi B, Marks AR, Bradman A, Harley K, Barr DB, Johnson C, Morga N, Jewell NP, 2007. Organophosphate pesticide exposure and neurodevelopment in young Mexican-American children. Environ. Health Perspect. 115, 792–798. 10.1289/ehp.9828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Gaines TB, 1969. Acute toxicity of pesticides. Toxicol. Appl. Pharmacol. 14, 515–534. 10.1016/0041-008x(69)90013-1. [DOI] [PubMed] [Google Scholar]
  24. Garcia SJ, Aschner M, Syversen T, 2006. Interspecies variation in toxicity of cholinesterase inhibitors. In: Gupta RC (Ed.), Toxicology of Organophosphate & Carbamate Compounds. Academic Press, Burlington, pp. 145–158. [Google Scholar]
  25. Johnson FO, Chambers JE, Nail CA, Givaruangsawat S, Carr RL, 2009. Developmental chlorpyrifos and methyl parathion exposure alters radial-arm maze performance in juvenile and adult rats. Toxicol. Sci. 109, 132–142. 10.1093/toxsci/kfp053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Johnson JA, Wallace KB, 1987. Species-related differences in the inhibition of brain acetylcholinesterase by paraoxon and malaoxon. Toxicol. Appl. Pharmacol. 88, 234–241. 10.1016/0041-008x(87)90009-3. [DOI] [PubMed] [Google Scholar]
  27. Levin ED, Timofeeva OA, Yang L, Petro A, Ryde IT, Wrench N, Seidler FJ, Slotkin TA, 2010. Early postnatal parathion exposure in rats causes sex-selective cognitive impairment and neurotransmitter defects which emerge in aging. Behav. Brain Res. 208, 319–327. 10.1016/j.bbr.2009.11.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Littell RC, Milliken GA, Stroup WW, Wolfinger RD, 1996. Sas System for Mixed Models 633. [Google Scholar]
  29. Maxwell DM, 1992. The specificity of carboxylesterase protection against the toxicity of organophosphorus compounds. Toxicol. Appl. Pharmacol. 114, 306–312. 10.1016/0041-008x(92)90082-4. [DOI] [PubMed] [Google Scholar]
  30. Meek EC, Carr RL, Chambers JE, 2021. In vitro age-related differences in rats to organophosphates. Toxicol. In Vitro 72, 105102. 10.1016/j.tiv.2021.105102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Moore S, Paalanen L, Melymuk L, Katsonouri A, Kolossa-Gehring M, Tolonen H, 2022. The association between ADHD and environmental chemicals-a scoping review. Int. J. Environ. Res. Public Health 19, 2849. 10.3390/ijerph19052849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Moser VC, Chanda SM, Mortensen SR, Padilla S, 1998. Age- and gender-related differences in sensitivity to chlorpyrifos in the rat reflect developmental profiles of esterase activities. Toxicol. Sci. 46, 211–222. 10.1006/toxs.1998.2526. [DOI] [PubMed] [Google Scholar]
  33. Perez-Fernandez C, Morales-Navas M, Aguilera-Saez LM, Abreu AC, Guardia-Escote L, Fernandez I, Garrido-Cardenas JA, Colomina MT, Gimenez E, Sanchez-Santed F, 2020a. Medium and long-term effects of low doses of chlorpyrifos during the postnatal, preweaning developmental stage on sociability, dominance, gut microbiota and plasma metabolites. Environ. Res. 184, 109341 10.1016/j.envres.2020.109341. [DOI] [PubMed] [Google Scholar]
  34. Perez-Fernandez C, Morales-Navas M, Guardia-Escote L, Colomina MT, Giménez E, Sánchez-Santed F, 2020b. Postnatal exposure to low doses of chlorpyrifos induces long-term effects on 5C-SRTT learning and performance, cholinergic and GABAergic systems and BDNF expression. Exp. Neurol. 330, 113356 10.1016/j.expneurol.2020.113356. [DOI] [PubMed] [Google Scholar]
  35. Poet TS, Wu H, Kousba AA, Timchalk C, 2003. In vitro rat hepatic and intestinal metabolism of the organophosphate pesticides chlorpyrifos and diazinon. Toxicol. Sci. 72, 193–200. 10.1093/toxsci/kfg035. [DOI] [PubMed] [Google Scholar]
  36. Rauh V, Arunajadai S, Horton M, Perera F, Hoepner L, Barr DB, Whyatt R, 2011. Seven-year neurodevelopmental scores and prenatal exposure to chlorpyrifos, a common agricultural pesticide. Environ. Health Perspect. 119, 1196–1201. 10.1289/ehp.1003160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rauh VA, Garfinkel R, Perera FP, Andrews HF, Hoepner L, Barr DB, Whitehead R, Tang D, Whyatt RW, 2006. Impact of prenatal chlorpyrifos exposure on neurodevelopment in the first 3 years of life among inner-city children. Pediatrics 118, e1845–e1859. 10.1542/peds.2006-0338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rauh VA, Perera FP, Horton MK, Whyatt RM, Bansal R, Hao X, Liu J, Barr DB, Slotkin TA, Peterson BS, 2012. Brain anomalies in children exposed prenatally to a common organophosphate pesticide. Proc. Natl. Acad. Sci. USA 109, 7871–7876. 10.1073/pnas.1203396109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ricceri L, Markina N, Valanzano A, Fortuna S, Cometa MF, Meneguz A, Calamandrei G, 2003. Developmental exposure to chlorpyrifos alters reactivity to environmental and social cues in adolescent mice. Toxicol. Appl. Pharmacol. 191, 189–201. 10.1016/s0041-008x(03)00229-1. [DOI] [PubMed] [Google Scholar]
  40. Roberts JR, Dawley EH, Reigart JR, 2019. Children’s low-level pesticide exposure and associations with autism and ADHD: a review. Pediatr. Res. 85, 234–241. 10.1038/s41390-018-0200-z. [DOI] [PubMed] [Google Scholar]
  41. Roegge CS, Timofeeva OA, Seidler FJ, Slotkin TA, Levin ED, 2008. Developmental diazinon neurotoxicity in rats: later effects on emotional response. Brain Res. Bull. 75, 166–172. 10.1016/j.brainresbull.2007.08.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Ross SM, McManus IC, Harrison V, Mason O, 2013. Neurobehavioral problems following low-level exposure to organophosphate pesticides: a systematic and meta-analytic review. Crit. Rev. Toxicol. 43, 21–44. 10.3109/10408444.2012.738645. [DOI] [PubMed] [Google Scholar]
  43. Rudakova EV, Boltneva NP, Makhaeva GF, 2011. Comparative analysis of esterase activities of human, mouse, and rat blood. Bull. Exp. Biol. Med. 152, 73–75. 10.1007/s10517-011-1457-y. [DOI] [PubMed] [Google Scholar]
  44. Storm JE, Rozman KK, Doull J, 2000. Occupational exposure limits for 30 organophosphate pesticides based on inhibition of red blood cell acetylcholinesterase. Toxicology 150, 1–29. 10.1016/s0300-483x(00)00219-5. [DOI] [PubMed] [Google Scholar]
  45. Tau GZ, Peterson BS, 2010. Normal development of brain circuits. Neuropsychopharmacology 35, 147–168. 10.1038/npp.2009.115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tessari L, Angriman M, Diaz-Roman A, Zhang J, Conca A, Cortese S, 2022. Association between exposure to pesticides and ADHD or autism spectrum disorder: a systematic review of the literature. J. Atten. Disord. 26, 48–71. 10.1177/1087054720940402. [DOI] [PubMed] [Google Scholar]
  47. Timofeeva OA, Roegge CS, Seidler FJ, Slotkin TA, Levin ED, 2008. Persistent cognitive alterations in rats after early postnatal exposure to low doses of the organophosphate pesticide, diazinon. Neurotoxicol. Teratol. 30, 38–45. 10.1016/j.ntt.2007.10.002. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Sette Supplement
NIHMS2107017-supplement-Sette_Supplement.docx (54.2KB, docx)

Data Availability Statement

Data will be made available on request.

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