Low level chlorpyrifos exposure increases anandamide accumulation in juvenile rat brain in the absence of brain cholinesterase inhibition

Abstract

The prevailing dogma is that chlorpyrifos (CPF) mediates its toxicity through inhibition of cholinesterase (ChE). However, in recent years, the toxicological effects of developmental CPF exposure have been attributed to an unknown non-cholinergic mechanism of action. We hypothesize that the endocannabinoid system may be an important target because of its vital role in nervous system development. We have previously reported that repeated exposure to CPF results in greater inhibition of fatty acid amide hydrolase (FAAH), the enzyme that metabolizes the endocannabinoid anandamide (AEA), than inhibition of either forebrain ChE or monoacylglycerol lipase (MAGL), the enzyme that metabolizes the endocannabinoid 2-arachidonylglycerol (2-AG). This exposure resulted in the accumulation of 2-AG and AEA in the forebrain of juvenile rats; however, even at the lowest dosage level used (1.0mg/kg), forebrain ChE inhibition was still present. Thus, it is not clear if FAAH activity would be inhibited at dosage levels that do not inhibit ChE. To determine this, 10 day old rat pups were exposed daily for 7 days to either corn oil or 0.5mg/kg CPF by oral gavage. At 4 and 12h post-exposure on the last day of administration, the activities of serum ChE and carboxylesterase (CES) and forebrain ChE, MAGL, and FAAH were determined as well as the forebrain AEA and 2-AG levels. Significant inhibition of serum ChE and CES was present at both 4 and 12h. There was no significant inhibition of the activities of forebrain ChE or MAGL and no significant change in the amount of 2-AG at either time point. On the other hand, while no statistically significant effects were observed at 4h, FAAH activity was significantly inhibited at 12h resulting in a significant accumulation of AEA. Although it is not clear if this level of accumulation impacts brain maturation, this study demonstrates that developmental CPF exposure at a level that does not inhibit brain ChE can alter components of endocannabinoid signaling.

Keywords: Chlorpyrifos; Developmental neurotoxicity; Endocannabinoid; Organophosphorus.

Figure 1

Chemical structures of the two major endocannabinoids anandamide and 2-arachidonoylglycerol.

Figure 2

Rates of weight gain of rat pups exposed daily from postnatal day 10 through 16 to either corn oil (control) or 0.5 mg/kg chlorpyrifos (CPS). Values are expressed as weight ± SE (n = 27–29).

Figure 3

Specific activity of (A) carboxylesterase (CES) and (B) cholinesterase (ChE) in the serum of rat pups exposed daily from postnatal day 10 through 16 to either corn oil (control) or 0.5 mg/kg chlorpyrifos (CPS). Values are expressed as mean ± SE (n = 12–17). Percent inhibition for each treatment group as compared to its respective control is presented in the oval overlaying the corresponding bar. Bars indicated with an asterisk (*) are statistically significant (p ≤ 0.05) from control.

Figure 4

Specific activity of (A) cholinesterase (ChE), (B) monoacylglycerol lipase (MAGL), and (C) fatty acid amide hydrolase (FAAH) in the forebrain of rat pups exposed daily from postnatal day 10 through 16 to either corn oil (control) or 0.5 mg/kg chlorpyrifos (CPF). Values are expressed as mean ± SE (n = 12–17). Percent inhibition for each treatment group as compared to its respective control is presented in the oval overlaying the corresponding bar. Bars indicated with an asterisk (*) are statistically significant (p ≤ 0.05) from control.

Figure 5

Levels of (A) 2-arachidonylglycerol (2-AG) and (B) arachidonoylethanolamide (AEA) in the forebrain of rat pups exposed daily from postnatal day 10 through 16 to either corn oil (control) or 0.5 mg/kg chlorpyrifos (CPF). Values are expressed as nmoles/g tissue ± SE for 2-AG and pmoles/g tissue ± SE for AEA (n = 12–17). Percent increase for each treatment group as compared to its respective control is presented in the oval overlaying the corresponding bar. Bars indicated with an asterisk (*) are statistically significant (p ≤ 0.05) from control.