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

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.

Effects of Chlorpyrifos on Serine Hydrolase Activities, Lipid Mediators, and Immune Responses in Lungs of Neonatal and Adult Mice.

Chlorpyrifos (CPF) is an organophosphate (OP) pesticide that causes acute toxicity by inhibiting acetylcholinesterase (AChE) in the nervous system. However, endocannabinoid (eCB) metabolizing enzymes in brain of neonatal rats are more sensitive than AChE to inhibition by CPF, leading to increased levels of eCBs. Because eCBs are immunomodulatory molecules, we investigated the association between eCB metabolism, lipid mediators, and immune function in adult and neonatal mice exposed to CPF. We focused on lung effects because epidemiologic studies have linked pesticide exposures to respiratory diseases. CPF was hypothesized to disrupt lung eCB metabolism and alter lung immune responses to lipopolysaccharide (LPS), and these effects would be more pronounced in neonatal mice due to an immature immune system. We first assessed the biochemical effects of CPF in adult mice (≥8 weeks old) and neonatal mice after administering CPF (2.5 mg/kg, oral) or vehicle for 7 days. Tissues were harvested 4 h after the last CPF treatment and lung microsomes from both age groups demonstrated CPF-dependent inhibition of carboxylesterases (Ces), a family of xenobiotic and lipid metabolizing enzymes, whereas AChE activity was inhibited in adult lungs only. Activity-based protein profiling (ABPP)-mass spectrometry of lung microsomes identified 31 and 32 individual serine hydrolases in neonatal lung and adult lung, respectively. Of these, Ces1c/Ces1d/Ces1b isoforms were partially inactivated by CPF in neonatal lung, whereas Ces1c/Ces1b and Ces1c/BChE were partially inactivated in adult female and male lungs, respectively, suggesting age- and sex-related differences in their sensitivity to CPF. Monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) activities in lung were unaffected by CPF. When LPS (1.25 mg/kg, i.p.) was administered following the 7-day CPF dosing period, little to no differences in lung immune responses (cytokines and immunophenotyping) were noted between the CPF and vehicle groups. However, a CPF-dependent increase in the amounts of dendritic cells and certain lipid mediators in female lung following LPS challenge was observed. Experiments in neonatal and adult Ces1d-/- mice yielded similar results as wild type mice (WT) following CPF treatment, except that CPF augmented LPS-induced Tnfa mRNA in adult Ces1d-/- mouse lungs. This effect was associated with decreased expression of Ces1c mRNA in Ces1d-/- mice versus WT mice in the setting of LPS exposure. We conclude that CPF exposure inactivates several Ces isoforms in mouse lung and, during an inflammatory response, increases certain lipid mediators in a female-dependent manner. However, it did not cause widespread altered lung immune effects in response to an LPS challenge.