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

Abstract

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.

Carboxylesterase 1d Inactivation Augments Lung Inflammation in Mice

“…Ces1d is expressed in mouse alveolar macrophages . In vitro experiments with these cells showed that baseline and LPS-induced levels of PGE 2 and cytokine mRNA (Il6 and TNFa) were higher in alveolar macrophages from Ces1d –/– mice compared to those from WT mice (Figure A).…”

“…Six or twenty-four hours after LPS injection, mice were sacrificed by exsanguination under isoflurane anesthesia (Figure A depicts the experimental protocol). The LPS dose and time points were the same as those in our previous studies. , Blood was collected from the inferior vena cava prior to exsanguination, and serum was prepared by centrifuging the clotted blood (4 °C, 10 min, 3,000 g ). Tissues (lung, liver, and adipose) were immediately flash-frozen in liquid nitrogen and stored at −80 °C until use.…”

“…Using activity-based protein profiling (ABPP) methods developed by Cravatt and co-workers, Ces1d was established to be the most active serine hydrolase in C57BL/6 mouse lung, followed by Ces1c . Previous research also suggested that the Ces/CES enzymes in alveolar macrophages and parenchymal lung tissues are involved in pesticide detoxication. ,− For example, exposures of mice to chlorpyrifos (CPF), an organophosphate pesticide, inhibited both Ces1c and Ces1d in neonate lung and Ces1c in adult lung due to stoichiometric covalent binding and degradation of the active metabolite chlorpyrifos oxon (CPO) . Although the lipopolysaccharide (LPS)-evoked pro-inflammatory cytokine levels in adult mouse lung were not altered by CPF exposure, CPF-treated females had augmented levels of LPS-induced eicosanoids (PGE 2 , PGD 2 , and 12-HETE).…”

“…Interestingly, inflammation can also influence the extent of Ces expression. For example, treatment of isolated mouse alveolar macrophages with LPS caused Ces1d mRNA levels to be decreased, suggesting that inflammatory pathways can modulate Ces1d expression. Similar attenuating effects of LPS on Ces2g mRNA and Ces2g activity levels in mouse spleen were observed in vivo .…”

“…Two types of lipid mediators involved in cell signaling that are degraded by CES1 in human monocytes/macrophages are 2-arachidonoylglycerol (2-AG), an endocannabinoid produced on demand in activated cells, and prostaglandin glyceryl esters (PG-Gs), which are cyclooxygenase-derived oxygenated metabolites of 2-AG . Although inhibition of CES1 was shown to increase the levels of anti-inflammatory 2-AG in human THP-1 monocytes and macrophages, monoacylglycerol lipase (Magl) has a more important role than Ces1d in 2-AG turnover in murine lung . In addition, small-molecule inhibitors that target CES1 have only a minor effect on 2-AG hydrolytic activity in human peripheral blood mononuclear cells, whereas Magl inhibitors have a major one. ,, (Patho)­physiological conditions that augment 2-AG levels in vivo, which is often referred to as increased ‘endocannabinoid tone,’ can modulate the degree of inflammation in tissue injury due to autocrine and paracrine signaling evoked by 2-AG .…”