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. Author manuscript; available in PMC: 2023 Apr 1.
Published in final edited form as: Toxicol In Vitro. 2022 Feb 11;80:105329. doi: 10.1016/j.tiv.2022.105329

Effects of chlorpyrifos on non-cholinergic toxicity endpoints in immortalized and primary rat hepatocytes under normal and hepatosteatotic conditions

SandeepReddy Kondakala 1, Lucie Henein 1, Erin McDevitt 1, Matthew K Ross 1, George Eli Howell III 1,*
PMCID: PMC8944201  NIHMSID: NIHMS1781203  PMID: 35151815

Abstract

Chlorpyrifos (CPS) is the most widely used organophosphate (OP) insecticide. Non-cholinergic targets of OPs include enzymes belonging to the serine hydrolase family. Carboxylesterases (Ces) are involved in detoxication of xenobiotics as well as lipid metabolism in the liver. Monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) are responsible for hydrolyzing endocannabinoids and can also be inhibited by OP compounds. However, there are no in vitro studies examining the sensitivities of these non-cholinergic endpoints following CPS exposure in the steatotic liver. Therefore, we determined the effects of CPS on these endpoints in immortalized McArdle-RH7777 (MCA) hepatoma cells and primary rat hepatocytes under normal and steatotic conditions. Ces activity was more sensitive to inhibition than MAGL or FAAH activity following exposure to the lowest CPS concentration. Additionally, Ces and MAGL activities in steatotic primary hepatocytes were less sensitive to CPS mediated inhibition than those in normal primary hepatocytes, whereas Ces inhibition was more pronounced in steatotic MCA cells. These findings suggest that steatotic conditions enhance the inhibition of hepatic serine hydrolases following exposure to CPS in an enzyme- and cell type-specific manner. CPS-mediated inhibition of these enzymes may play a part in the alterations of hepatic lipid metabolism following OP exposures.

Keywords: organophosphate, chlorpyrifos, hepatosteatosis, carboxylesterase, endocannabinoid system

2. Introduction:

Chlorpyrifos (CPS) is an organophosphorus (OP) insecticide that is widely used to control pest infestations. It is extensively used for agriculture purposes but was banned in 2000 for household purposes (Solomon et al., 2014). The phosphorothionates, including CPS, are bioactivated by desulfuration to the active oxon form in the liver by cytochrome P450s (Sultatos et al., 1984). These oxon metabolites are potent and irreversible inhibitors of acetylcholinesterase (AChE). AChE belongs to the serine hydrolase family of enzymes, is involved in the hydrolysis of the neurotransmitter acetylcholine, and its inhibition leads to an increase in acetylcholine levels at the synapse resulting in overstimulation within the central and peripheral nervous systems (Chambers and Carr, 1993).

Although the oxon metabolite of CPS, chlorpyrifos-oxon (CPO), is considered primarily neurotoxic, it can also produce effects in the periphery by inhibiting other serine hydrolase enzymes resulting in potential non-cholinergic effects by targeting monoacylglycerol lipase (MAGL), fatty acid amide hydrolase (FAAH), and carboxylesterase (Ces) (Casida and Quistad, 2005). Ces enzymes belong to the serine hydrolase superfamily and react with oxon metabolites via covalent interaction that inactivate these molecules (Sogorb and Vilanova, 2002). They also catalyze the hydrolytic metabolism of several ester-containing xenobiotics including drugs and pollutants (Ross et al., 2010; Sogorb and Vilanova, 2002). Ces enzymes are highly expressed in the liver (Ko et al., 2009). In addition to their important roles in xenobiotic metabolism, Ces enzymes are also involved in the hydrolysis of 2-arachidonoylglycerol (2-AG) and hepatic triacylglycerol metabolism (Quiroga et al., 2012; Xie et al., 2010). MAGL and FAAH enzymes are a part of the endocannabinoid system, which consists of the endocannabinoid receptors cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2), as well as their endogenous endocannabinoid ligands.

The most widely studied endocannabinoids (ECBs) are the two main lipid mediators 2-AG and anandamide (AEA), which are also produced in the liver (Di Marzo, 2008). Further, CB1 receptors are present in peripheral organs, such as the liver, and the central nervous system, whereas CB2 receptors are mainly present in immune cells (Di Marzo, 2008). Hepatic CB1 expression is low in healthy liver, but their expression levels are increased during altered physiological conditions such as hepatic steatosis and obesity (Osei-Hyiaman et al., 2005; Osei-Hyiaman et al., 2008). The activation of CB1 receptors stimulates fatty acid synthesis and de novo lipogenesis, and it disrupts insulin receptor signaling in the liver (Chanda et al., 2012). The principal catabolic enzymes of the ECBs are MAGL and FAAH, which release arachidonic acid (AA) in the process. 2-AG is primarily hydrolyzed by MAGL, which is highly expressed in liver, brain, and white adipose tissue. However, 2-AG is also hydrolyzed by FAAH, ABHD6, and Ces enzymes (Blankman et al., 2007; Xie et al., 2010). On the other hand, AEA seems to be hydrolyzed specifically by FAAH, which is highly expressed in both the liver and brain (Quistad et al., 2001).

Several in vivo rodent studies have determined that exposure to OP pesticides, including CPS, under normal metabolic conditions caused the inhibition of Ces, FAAH, and MAGL in the brain and liver (Carr et al., 2011; Casida and Quistad, 2004; Howell III et al., 2018; Kondakala et al., 2017; Quistad et al., 2006a). However, no in vitro studies have explored the potency of CPS on these non-cholinergic targets in hepatocytes under normal or induced steatotic conditions. Thus, the goal of the present study was to determine the in vitro effects of direct exposure to CPS on non-cholinergic endpoints of OP toxicity in the liver using cell culture models. The current study utilized immortalized rat McA-RH7777 (MCA) hepatoma cells and primary rat hepatocytes as in vitro hepatocyte models. Both immortalized and primary cells were used to determine if there was a difference between a widely used immortalized cell model and a freshly isolated primary cell model.

3. Materials and Methods:

3.1. Chemicals:

CPS (≥99% purity) was purchased from Sigma-Aldrich (St. Louis, MO) and CPS stock solutions were prepared in dimethyl sulfoxide (DMSO). CPO (99% purity) was purchased from ChemService (West Chester, PA). AChE from Electrophorus electricus (electric eel), glucose-6-phosphate dehydrogenase (G6PDH), β-nicotinamide adenine dinucleotide phosphate (NADP), and glucose-6-phosphate (G6P) were purchased from Sigma-Aldrich (St. Louis, MO). Oleic acid (OA) and palmitic acid (PA) fatty acid stocks were prepared in fatty acid-free 10% (w/v) bovine serum albumin (BSA; Sigma-Aldrich, MO). 2-AG (≥95% purity), AEA (≥98% purity), and arachidonic acid-d8 (AA-d8, ≥96% purity) were purchased from Cayman Chemicals (Ann Arbor, MI). The mobile phase solvents used for the ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) analysis were methanol (Optima ® LC/MS grade), water (Optima ® LC/MS grade), and acetonitrile (Optima ® LC/MS grade) purchased from Fisher Chemical (Thermo Fisher Scientific, Waltham, MA). The media and supplements used for cell culture consisted of Dulbecco’s Modified Eagle’s Medium (DMEM), Roswell Park Memorial Institute (RPMI 1640) media, fetal bovine serum (FBS), sodium pyruvate, penicillin, and streptomycin and were purchased from Sigma-Aldrich (St. Louis, MO).

3.2. Immortalized cell culture:

MCA hepatoma cells were obtained from American Type Culture Collection (ATCC) and cultured as previously performed (Howell et al., 2016). Briefly, MCA cells were grown in DMEM with high glucose (4500 mg/L) containing glutamine (4 mM), sodium pyruvate (1%), penicillin-streptomycin (1%), and 20% FBS. MCA cells were maintained in an incubator at 37°C with 5% CO2 and passaged continuously when 70% confluent. MCA cells were plated in rat tail collagen (0.025%) coated plates at a density of 5×105 cells/ml and incubated overnight to allow for attachment to the surface prior to starting treatments.

3.3. Primary hepatocytes:

Primary hepatocytes were isolated from male Sprague-Dawley rats as previously performed (Howell et al., 2009; Howell et al., 2018a). Briefly, each rat was anesthetized with a continuous flow of oxygen-containing isoflurane (2–5%). The abdomen was cleaned with ethanol and cut through the midline of the abdomen to expose the hepatic portal vein. A blunt end cannula was then inserted in the portal vein and the liver was flushed with Hank’s Balanced Salt Solution (HBSS). The liver was then perfused with HBSS (1X) containing collagenase (Type I 100 U/ml) until the liver began to digest, as evident by a “honeycomb” type appearance due to the breakdown of connective tissue. The liver was then removed from the animal, minced, and placed in a shaking water bath (37°C) for 10 minutes. The liver homogenate was passed through a sterile filter (100 microns) and centrifuged (100 × g, 2 minutes, 4°C) to pellet the hepatocytes. The pellet was then resuspended in media and passed through another sterile filter (70 μm). To further purify the hepatocytes, the filtrate was then centrifuged (100 × g, 1 minute, 4°C) to form a pellet, resuspended again in media, and centrifuged (100 × g, 1 minute, 4°C). This process was repeated two more times to further purify the hepatocyte fraction. The isolated hepatocytes were then subjected to trypan blue staining to determine cell viability via counting with a hemocytometer.

Primary hepatocytes were plated in rat tail collagen (0.025%) coated 100 mm petri dishes or 6-well plates at a density of 1×106 cells/ml in growth media consisting of RPMI 1640 supplemented with 10% FBS, glucose (20 mM), insulin (100 nM), penicillin and streptomycin. After plating, the cells were incubated for 3–4 hours to allow them to attach to the surface of the plate before starting treatments. Prior to experimental use, hepatocytes were washed with phosphate buffered saline (PBS; pH 7.4) to remove unattached cells and the media was changed to treatment media containing RPMI 1640 with BSA (0.5%) and no FBS for 24 hours. FBS was not added to the treatment media to negate any confounding effects of potential CPS degradation from FBS components.

3.4. Experimental design:

To determine the concentration-dependent effects of CPS, MCA cells and primary hepatocytes were exposed to CPS (0.002 μM, 0.02 μM, 0.2 μM, 2 μM, 20 μM) for either 8 or 24 hours or to vehicle (DMSO; 0.025%) or control media (without CPS or DMSO). It should be noted that the DMSO vehicle did not have any effect on the analyzed endpoints and thus the data from the control media groups are not shown. The selected CPS concentrations utilized in this study are based on previous studies from our lab and by others. Previous studies from our lab indicate that exposure to CPS (at ≥ 5 μM) produced significant increases in neutral lipid accumulation with no cytotoxic effects (Howell et al., 2016). The current low end concentrations are comparable to a previous study in hepatocytes where exposure to CPS (0.1 μM, 1 μM, and 10 μM) induced the levels of CYP3A4, which is also a major CYP isoform involved in the bioactivation of CPS (Rouimi et al., 2012). Additionally, the concentration range of CPS in this study is comparable to the observed serum concentrations of CPS in cases of self-poisoning in humans following acute CPS exposure where the peak serum concentrations are in the low micromolar (<10 μM) range (Eddleston et al., 2005; Eyer et al., 2009).

The cultured cells were exposed to exogenous fatty acids prior to the CPS treatments to induce steatotic conditions and to enable the effect of this condition on CPS toxicity to be determined. In this study, we utilized a 2:1 molar ratio of OA (100 μM or 300 μM) and PA (50 μM or 150 μM) in media containing 1% BSA and treated the cells for 24 hours. OA and PA are the two major fatty acids that are most abundant in western diets and are typically present in a 2:1 molar ratio. The fatty acid concentrations used in this study are based on previous studies that used these fatty acids to induce lipid accumulation and steatotic conditions in hepatocytes (Howell et al., 2018b; Howell et al., 2016; Ricchi et al., 2009). Following 24 hours of fatty acid exposure, cells were treated with media containing BSA (1%) only or vehicle (0.025% DMSO) only or CPS (0.002 μM, 0.02 μM, 0.2 μM, 2 μM) for 8 hours in serum-free treatment media. After the designated exposure duration, cell monolayers were washed with PBS, cells were scraped and homogenates were made by sonication in 50 mM Tris-HCl buffer (pH 7.4) for Ces, MAGL, and FAAH activity assays.

3.5. Lipid accumulation determined by Oil Red O (ORO) staining:

Following exposures, culture media was removed, and the cells were fixed with 10% buffered formalin for at least 30 minutes. Following fixation, cells were washed with 60% isopropanol and ORO stain (100 μl per well) was added for 15 minutes at room temperature. Cells were washed 4 times with deionized water and allowed to air dry. Intracellular ORO stain was extracted from the cells with 100% isopropanol and the absorbance (520 nm) of the solution was measured to determine the amount of ORO. To normalize for cell number following ORO staining, cell monolayers were stained with Janus Green for 5 minutes. Janus Green was extracted from the cells with 0.5 N HCl and the absorbance was measured at 595 nm. The neutral lipid accumulation was expressed as the ratio of ORO absorbance (520 nm) to Janus Green absorbance (595 nm) as previously performed (Howell et al., 2018a, b; Howell et al., 2016).

3.6. Ces activity assay:

Following the CPS treatments, cell lysates were homogenized according to the procedure previously described (Ross and Borazjani, 2007). Briefly, cell lysates were diluted to 50 μg protein/ml (final concentration) in 50 mM Tris-HCl buffer, pH 7.4 (150 μl final volume) and pre-incubated for 5 minutes at 37°C. The substrate para-nitrophenyl valerate (p-npv; final concentration of 500 μM) was then added to initiate the reaction. The reaction was measured at 405 nm with 20 seconds intervals over 5 minutes to determine the rate of formation for para-nitrophenol. The slopes of the activity curves (absorbance units per minute) were calculated by using an extinction coefficient of 13 cm−1 mM−1 then data were normalized to enzyme activity units and expressed as μmol minute (min−1) mg protein (P−1) (Morgan et al., 1994; Ross and Borazjani, 2007).

3.7. MAGL and FAAH activity in cell lysates:

Following the treatments, cell lysates were made as described above. Cell lysate homogenates were diluted to the final protein concentration of 0.5 mg/ml in 50 mM Tris-HCl buffer (pH 7.4) to a total reaction volume (100 μl) and pre-incubated for 5 minutes at 37°C. Following pre-incubation, samples were fortified with 2-AG (50 μM) or AEA (50 μM) or 2% v/v ethanol (2 μl) as a background control and incubated for 30 minutes at 37°C. The reactions were terminated with cold acetonitrile containing AA-d8 (2.5 μM) and incubated for 10 minutes on ice. Samples were then centrifuged (10 minutes,10,000 × g, 4°C) and the supernatant was transferred to LC vials for UPLC-MS/MS analysis of AA levels in the homogenates (Howell III et al., 2018).

AA analysis was performed by UPLC-MS/MS. The mobile phases used were a blend of solvent A (0.1% v/v acetic acid in water) and solvent B (0.1% v/v acetic acid in acetonitrile). Samples (20 μl) were injected onto an Acquity UPLC BEH C18 column (1.7 μm, 100×2.1 mm I.D.) equipped with a guard column. The following gradient program was set at a flow rate of 0.2 ml/min for 20 min and the analytes were eluted with a linear gradient of solvent B (5% to 95% B between 1–10 min, held at 95% B for 5 min, then returned to 5% B and the column re-equilibrated for 5 min). The column eluate was directed into the mass spectrometer via heated electrospray ionization in negative ion mode. AA and its deuterated standard were observed by single reaction monitoring (SRM) according to these settings: AA, [M-H]− m/z 303.3 > 259.5; AA-d8, [M-H]− m/z 311.3 > 267.5. Scan times were 0.2 seconds per SRM and the scan width was 0.01 m/z. AA levels were quantified by measuring the area under each chromatographic peak and comparing it to the area under the chromatographic peak for the deuterated internal standard, followed by correction for the instrument response factor of AA relative to its deuterated standard (determined using a calibration curve) to yield absolute amounts of AA. In general, the data were expressed as a simple ratio of AA peak area/AA-d8 peak area (Howell III et al., 2018; Kondakala et al., 2017).

3.8. CPS bioactivation assay:

Bioactivation of CPS by P450 enzyme activity was determined by a desulfuration assay as previously described using hepatocyte microsomes (Kondakala et al., 2017). Preparation of cell microsomes and assay for CPS desulfuration were performed as previously described with modifications (Chambers and Chambers, 1989). Following the treatments, cells were washed with PBS (pH 7.4) buffer and homogenized in 3 ml of 50 mM Tris-HCl (pH 7.4). Cells were sonicated for 10 seconds 3 times and then centrifuged (17,000 × g, 15 minutes, 4°C). The collected supernatant was centrifuged (100,000 × g, 1 hour, 4°C) again to obtain the microsomal pellet, covered with glycerol storage buffer (20% v/v glycerol, 0.2 mM potassium EDTA, 0.1 mM potassium phosphate buffer, 0.5 mM dithiothreitol), and stored at −80°C until further analysis. For the assay, microsomal pellets (0.2 μg/ml) were diluted in 50 mM Tris-HCl (pH 7.4) buffer containing 5 mM MgCl2 in a total reaction volume of 2 ml. The formation of CPO was measured by monitoring the inhibition of the external source of AChE which was from the electric eel. Eel AChE (0.2 U/ml, final concentration) was added to the reaction with the NADPH-generating system, which consisted of NADP (2.47 mg/ml), G6P (78.75 mg/ml) and G6PDH (5 U/ml). This mixture was incubated with the microsomes at 37°C for 30 minutes. CPS (80 μM) was included as the substrate with the controls being ethanol (5 μl) and eserine sulfate (10 μM). Then 1.75 ml of 50 mM Tris-HCl (pH 7.4) buffer with acetylthiocholine (ATCh, 1 mM) was added to initiate the reaction and incubated for 15 minutes at 37°C. The reactions were stopped by adding a 4:1 (v/v) mixture of sodium dodecyl sulfate:5,5-dithio-bis-(2-nitrobenzoic acid) (SDS 0.5%:DTNB 0.0024 M) at 10 second intervals, and absorbance was read spectrophotometrically at 412 nm.

CPO generated in the hepatocyte microsomes was quantified from a standard curve of CPO versus electric eel AChE inhibition. A range of CPO concentrations (1 × 10−5 M to 1 × 10−7 M) were used to result in AChE inhibition between 10% and 90%. Linear regression analysis was executed with the plot of the logit of percent inhibition versus log10 oxon concentration. The best-fit line was drawn using points corresponding to the 10–90% AChE inhibition range. The standard curve was prepared three independent times using a unique set of reagents, and the data were averaged to produce a single standard curve to use for the quantification of CPS bioactivation to CPO. Data expressed as CPO nmol min−1 mg P−1 (Kondakala et al., 2017).

3.9. Statistical analysis:

All data in the treatment and control groups are presented as the mean ± standard error of the mean (SEM). Statistical analysis of 2-AG and AEA hydrolysis assays was performed on transformed (log10 transformation) AA/AA-d8 ratios. The one-way analysis of variance (ANOVA) with Tukey’s post hoc test for multiple pairwise comparisons with a confidence interval of 95 percent was used to analyze the data for CPS concentration dependent inhibition of enzyme activities. The statistical analysis for the data for steatotic or normal conditions was analyzed by two-way ANOVA with Tukey’s post hoc test using multiple comparisons to determine the effects of fatty acid exposure (steatosis) and concentration of CPS on enzymatic activities. In experiments that compared two groups, a Student’s t-test was used. A P-value less than or equal to 0.05 (P<0.05) was used to indicate a statistical difference between groups.

4. Results:

4.1. Effect of CPS on Ces activity under normal cell culture conditions:

Exposure to CPS resulted in both time and concentration dependent decreases in overall Ces activity in both MCA cells and rat primary hepatocytes (Figure 1). Regarding effects on Ces activity in MCA cells, exposure to CPS for 8 hours (Figure 1A) significantly decreased Ces activity at lowest concentration utilized (0.002 μM) compared to vehicle (0 μM) and this decrease was more pronounced with increasing concentrations of CPS (0.02 – 20 μM). The IC50 for CPS mediated Ces inhibition in MCA cells following 8 hours of exposure was 3.3 nM (Supplemental table 1). CPS mediated decreases in Ces activity in MCA cells were greater following exposure for 24 hours (Figure 1B) with the lowest concentration of CPS (0.002 μM) producing approximately twice the amount of inhibition of Ces compared to the 8-hour time point. However, there were similar reductions in Ces activity following 8 and 24 hours of exposure to the highest concentration of CPS (20 μM). The IC50 value for CPS mediated Ces inhibition in MCA cells following 24 hours of CPS exposure was not able to be calculated.

Figure 1:

Figure 1:

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Ces activities following exposure to CPS for 8 or 24 hours in both MCA cells and primary hepatocytes. Data represent the mean ± SEM of 4–6 independent replicates for MCA cells (A and B) and 4–5 animals for primary hepatocytes (C and D). Groups with the same letter are not significantly different (P<0.05).

As opposed to MCA cells, Ces activity in primary hepatocytes was less sensitive to CPS mediated inhibition and this inhibition was not as time sensitive as in the MCA cells. Ces activity in primary hepatocytes was significantly inhibited following exposure to CPS (0.2 μM) for 8 (Figure 1C) and 24 hours (Figure 1D) with maximal reductions in Ces activity following exposure to CPS (20 μM). The IC50 values for CPS mediated inhibition of Ces in primary hepatocytes was approximately 150 nM and 180 nM at 8 and 24 hours, respectively (Supplemental table 2).

4.2. Effect of CPS on FAAH activity under normal conditions:

Exposure to CPS in MCA cells produced both time and concentration dependent inhibition of FAAH activity similar to that observed for Ces activity. Following exposure for 8 hours, FAAH activity was significantly inhibited by exposure to CPS (0.02 μM) and this suppression in activity was comparable to the inhibition produced by the highest concentration of CPS (Figure 2A). The IC50 for CPS mediated FAAH inhibition following 8 hours of exposure was 5.2 nM (Supplemental table 1). CPS mediated inhibition of FAAH was more pronounced following exposure for 24 hours with the lowest concentration of CPS (0.002 μM) producing significant reductions in FAAH activity and these reductions persisted through the highest concentration of CPS (Figure 2B). The IC50 for CPS mediated FAAH inhibition following 24 hours of exposure was 0.7 nM (Supplemental table 1).

Figure 2:

Figure 2:

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FAAH activities following exposure to CPS for 8 or 24 hours in both MCA cells and primary hepatocytes. Data represent the mean ± SEM of 4–6 independent replicates for MCA cells (A and B) and 4–5 animals for primary hepatocytes (C and D). Statistical analysis was performed on log10 transformed values. Groups with the same letter are not significantly different (P<0.05).

As observed with Ces activity, FAAH activity in primary hepatocytes was less sensitive to inhibition following CPS exposure than FAAH activity in MCA cells. FAAH activity was significantly inhibited following exposure to CPS (0.2 μM) for 8 hours and there was a concentration dependent decrease in FAAH activity (Figure 2C). The IC50 for CPS mediated FAAH inhibition following 8 hours of exposure was approximately 960 nM (Supplemental table 1). However, only the highest concentration of CPS (20 μM) produced significant inhibition of FAAH activity following exposure for 24 hours. The IC50 value for CPS mediated FAAH inhibition in primary hepatocytes following 24 hours of CPS exposure was not able to be calculated.

4.3. Effect of CPS on MAGL activity under normal conditions:

MAGL activity was not as sensitive as Ces and FAAH activities in MCA cells to CPS mediated inhibition. Significant reductions in MAGL activities were observed following exposure to CPS (0.2 μM) for 8 hours and these reductions were similar to those observed following exposure to the highest concentration of CPS (Figure 3A). The IC50 for CPS mediated MAGL inhibition following 8 hours of CPS exposure was 17.9 nM (Supplemental table 1). Following exposure to CPS (0.002 μM) for 24 hours there was a significant reduction in MAGL activity in MCA cells which increased as the concentration of CPS increased (Figure 3B). The IC50 for CPS mediated MAGL inhibition following 24 hours of CPS exposure was 1.46 nM (Supplemental table 1).

Figure 3:

Figure 3:

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MAGL activities following exposure to CPS for 8 or 24 hours in both MCA cells and primary hepatocytes. Data represent the mean ± SEM of 4–6 independent replicates for MCA cells (A and B) and 4–5 animals for primary hepatocytes (C and D). Statistical analysis was performed on log10 transformed values. Groups with the same letter are not significantly different (P<0.05).

As observed with Ces and FAAH activities, MAGL activity in primary hepatocytes was not as sensitive to CPS mediated inhibition as it was in MCA cells. Only the highest concentration of CPS (20 μM) produced a significant reduction in MAGL activity following 8 hours of CPS exposure in primary hepatocytes (Figure 3C). A comparable reduction in MAGL activity was observed following 24 hours of CPS (20 μM) exposure (Figure 3D). The IC50 values for MAGL inhibition in primary hepatocytes at 8 and 24 hours of exposure were not able to be calculated.

4.4. CPS effect on neutral lipid accumulation under steatotic conditions:

Following exposures of hepatocytes to fatty acids and CPS for 8 hours, neutral lipid accumulation was determined by ORO staining (Figure 4). OA:PA (300 μM:150 μM) in MCA cells produced a significant increase in neutral lipid accumulation compared to control and vehicle treated cells (Figure 4A). However, CPS exposure did not produce any effect on the neutral lipid accumulation in MCA cells. OA:PA (300 μM:150 μM) also produced a significant increase in neutral lipid accumulation in primary hepatocytes but CPS exposure did not have any effect on lipid accumulation compared to control and vehicle treated normal cells (Figure 4B).

Figure 4:

Figure 4:

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Neutral lipid accumulation was determined with ORO staining in MCA cells and primary hepatocytes pretreated in serum free or media containing oleic acid (OA) and palmitic acid (PA) in a 2:1 ratio for 24 hours then exposed to CPS for 8 hours. Data represent the mean ± SEM of 7–9 independent replicates for MCA cells (A) and 5–6 animals for primary hepatocytes (B). Groups with the same letter are not significantly different (P<0.05).

4.5. Bioactivation capacity over the duration of exposures under normal or steatotic conditions:

The bioactivation of CPS in hepatocytes mediated by CYP450 was determined by in situ formation of CPO with the desulfuration assay in cell microsomes (Figure 5). The bioactivation capacity of cell microsomes was determined from cells collected at 0 hours, 24 hours, and 48 hours after seeding cells in culture dishes. The cells were exposed to culture media supplemented with or without fatty acids for the first 24 hours, then the fatty acid-containing media was replaced with normal treatment media for an additional 24 hours producing a maximal culture time of 48 hours. The bioactivation capacity in MCA and primary cells was decreased after 24 hours and 48 hours under normal conditions compared to the 0-hour starting time point, although the 48-hour time point for the primary cells did not reach statistical significance (Figure 5A and 5B). However, there was no significant difference between the 24- and 48-hour time points in MCA cells or primary hepatocytes, indicating no loss of bioactivation capacity between these time intervals. Additionally, fatty acid exposure did not significantly alter the bioactivation of CPS in MCA cells (Figure 5C) or primary hepatocytes (Figure 5D).

Figure 5:

Figure 5:

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Bioactivation capacity of MCA cells (A) and primary hepatocytes (B) during the duration of culture under normal conditions and effects of exposure to oleic acid (OA):palmitic acid (PA) on bioactivation capacity in MCA cells (C) and primary hepatocytes (D) for up to 48 hours in culture. Bioactivation capacity was determined by desulfuration assay in cellular microsomes. Data represent the mean ± SEM of 5 independent replicates for MCA cells and 5 animals for primary hepatocytes. Groups with the same letter are not significantly different (P<0.05).

4.6. Effect of steatotic conditions on CPS inhibition of Ces activity in steatotic conditions:

Following exposure to CPS in both normal conditions and steatotic conditions, Ces activity was determined in cell lysates (Figure 6). Steatotic MCA cells had a significant decrease in Ces activity following exposure to the lowest concentration of CPS (0.02 μM) and higher concentrations compared to the vehicle (Figure 6A). Normal (no fatty acid exposure) MCA cells had a significant decrease following exposure to CPS 0.2 μM and higher concentrations. Ces activity was significantly decreased within CPS treatment groups in steatotic MCA cells and showed greater sensitivity to CPS mediated inhibition of Ces compared to normal MCA cells. Exposure to CPS 2 μM in primary hepatocytes produced a significant decrease in Ces activity (Figure 6B). However, unlike in the MCA cells, there were no significant effects of steatotic conditions on CPS exposure mediated Ces inhibition in primary hepatocytes.

Figure 6:

Figure 6:

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Ces activity in lysates of normal and oleic:palmitic acid (OA:PA) treated cells following 8 hours of CPS exposure. Data represent the mean ± SEM of 4–5 independent replicates for MCA cells (A) and 5–6 animals for primary hepatocytes (B). Groups with the same letter are not significantly different (P<0.05).

4.7. Effect of steatotic conditions on CPS inhibition of FAAH activity:

Following CPS exposure in both normal and steatotic conditions, FAAH activity was significantly decreased following exposure to CPS 0.02 μM and higher concentrations in MCA cells (Figure 7A). However, primary hepatocytes were less sensitive to FAAH inhibition following CPS exposure compared to MCA cells. Exposure to CPS 2 μM in primary hepatocytes produced a significant decrease in both normal and steatotic conditions (Figure 7B). Thus, there were no significant effects of the steatotic conditions on CPS mediated inhibition of FAAH activity in MCA cells or primary hepatocytes.

Figure 7:

Figure 7:

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Hydrolysis of AEA in lysates of normal and oleic:palmitic acid (OA:PA) treated cells following 8 hours of CPS exposure as an index of FAAH activity. Data represent the mean ± SEM of 4–5 independent replicates for MCA cells (A) and 4–6 animals for primary hepatocytes (B). Statistical analysis was performed on log10 transformed values. Groups with the same letter are not significantly different (P<0.05).

4.8. Effect of steatotic conditions on CPS toxicity with MAGL activity:

MAGL activity was determined in cell lysates following exposure to CPS in normal or fatty acid induced steatotic conditions in both MCA cells and primary hepatocytes (Figure 8). Following CPS exposure for 8 hours, there was significant inhibition of MAGL activity following exposure to CPS 0.02 μM and higher concentrations in both normal and steatotic MCA cells (Figure 8A). There was no significant effect of steatotic conditions on MAGL activity following exposure to CPS in MCA cells. Following exposure to CPS 2 μM, there was a significant decrease in both normal and steatotic primary hepatocytes compared to lower concentrations of CPS (Figure 8B). However, steatotic primary hepatocytes were less sensitive to MAGL inhibition and as indicated by lower inhibition following exposure to CPS 2 μM when compared to normal cells.

Figure 8:

Figure 8:

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Hydrolysis of 2-AG in lysates of normal and oleic:palmitic acid (OA:PA) treated cells following 8 hours of CPS exposure as an index of MAGL activity. Data represent the mean ± SEM of 4–5 independent replicates for MCA cells (A) and 4–6 animals for primary hepatocytes (B). Statistical analysis was performed on log10 transformed values. Groups with the same letter are not significantly different (P<0.05).

5. Discussion:

To our knowledge, no in vitro studies have examined the effects of CPS exposure on non-cholinergic targets of OP toxicity under normal and induced steatotic conditions in immortalized or primary hepatocytes. Thus, this study is the first to examine the inhibition of Ces, FAAH, and MAGL activities in cultured hepatocytes following CPS exposure, and whether neutral lipid accumulation in hepatocytes alters the degree of enzyme inhibition. Exposure to OP insecticides, including CPS, has been associated with metabolic disease conditions such as hypertriglyceridemia and hepatic steatosis (Howell et al., 2016; Slotkin et al., 2005). These CPS-dependent effects on lipid metabolism may be due to the inhibition of non-cholinergic target endpoints. Hepatic steatosis is the main characteristic of NAFLD. This increase in neutral lipid accumulation within hepatocytes can occur due to an increase in circulating free fatty acids (FFA), which results in increased uptake of FFAs, and metabolic alterations including de novo lipogenesis (DNL), fatty acid oxidation, and VLDL secretion in the hepatocyte (Fabbrini et al., 2010). OA and PA are the major fatty acids available in western diets and in vitro studies have shown that utilization of the fatty acids in 2:1 ratio increased lipid accumulation in cultured hepatocytes (Ricchi et al., 2009). In our current study, we utilized OA:PA (300:150 μM) and incubated cells with this fatty acid mixture for 24 hours to induce steatotic conditions prior to CPS treatments. This approach increased neutral lipid accumulation in both MCA cells and primary hepatocytes, which is mainly due to the accumulation of triglycerides (Figure 4).

CPS is metabolized in the liver by different pathways including bioactivation and detoxication processes. It is well known that primary hepatocyte cultures lose their phase 1-associated CYP450 activity, so their application is limited to short-term studies (Donato et al., 1994). MCA cells are immortalized cells and used as an in vitro model to examine hepatocyte lipid metabolism because their lipoprotein metabolism and expression of molecular mediators of lipogenesis are similar to primary hepatocytes (Boren et al., 1994; Chamberlain et al., 2013). Therefore, in this current study, we utilized MCA cells and rat primary hepatocytes to examine the selective potency of CPS on targets of OP toxicity, the effect of steatotic conditions prior to exposure on CPS mediated toxicity, and the bioactivation of CPS (Donato et al., 1994; Holme, 1985). The metabolism of OP insecticides mainly relies on the cytochrome P450 system in hepatocytes for bioactivation or detoxication of insecticides (Tang et al., 2001). In the current study, the bioactivation capacity of CPS by microsomes of MCA and primary hepatocytes under normal conditions was significantly decreased after 24 hours in culture, whereas no further decreases were observed by 48 hours. Steatotic conditions in both MCA cells and primary hepatocytes did not show any significant effect on the bioactivation of CPS. This indicates that steatotic hepatocytes did not affect the formation of CPO (Figure 5).

Ces is highly expressed in the liver, and it is also present in rodent plasma but not human plasma (Ross and Borazjani, 2007; Satoh and Hosokawa, 1998). One of the major functions of Ces is the metabolism of xenobiotics, including the inactivation of OP oxon metabolites such as CPO. However, it also has important roles in lipid metabolism and VLDL secretion from hepatocytes (Lehner et al., 2012). Crow and co-workers demonstrated that recombinant human CES1 and CES2 isoforms were potently inhibited by CPO (IC50s, 0.15 nM and 0.33 nM for CES1 and CES2, respectively) (Crow et al., 2012). In the current study, the IC50 of Ces activity in MCA cells following 8 hours of CPS exposure was 3.3 nM, whereas in primary hepatocytes it was 150 nM (Supplemental Tables 1 and 2). The discrepancy in IC50s in the current study and those in Crow et al. (2012) might be due to species differences between the Ces isoforms, although a more likely possibility is that the current study employed an intact cell model in which CPS must be converted to CPO prior to Ces inhibition.

The physiological actions of the endocannabinoids 2-AG and AEA are terminated by their hydrolysis, primarily by MAGL and FAAH, respectively. Although MAGL is the major hydrolytic enzyme of 2-AG, it can also be hydrolyzed by Ces, FAAH and ABHD6 (Carr et al., 2011; Di Marzo, 2008). Earlier studies demonstrated that OP oxon metabolites, including CPO, inhibited the ECB metabolizing enzymes FAAH and MAGL in brain homogenates and in brains of rodents following in vivo exposures (Nomura et al., 2008; Quistad et al., 2006a; Quistad et al., 2006b; Quistad et al., 2001). The IC50s for CPO-dependent inhibition of FAAH and MAGL activity in mouse brain homogenates was 40 nM and 34 nM, respectively (Quistad et al., 2006a; Quistad et al., 2001). Further, the inhibition of FAAH and MAGL following CPS exposure in rats resulted in elevated ECB levels in brain (Carr et al., 2011). Previous studies demonstrated that treatment of HepG2 cells with agonists of CB1 and CB2 increased the degree of OA-induced steatosis in a concentration dependent manner (De Gottardi et al., 2010). In the current study, CPS exposure caused significant inhibition of MAGL and FAAH after 8 and 24 hours, which may lead to an increase in ECB levels. It is, therefore, possible that an increase in ECB levels could increase the activation of CB1 receptors and, in turn, increase DNL. Taken together, steatotic conditions did not produce a significant effect on CPS-mediated inhibition of FAAH activity in either MCA cells or primary hepatocytes. MAGL was less sensitive to inhibition in steatotic primary hepatocytes than in steatotic MCA cells. The disruption of FAAH and MAGL activities following CPS exposures may increase the severity of the steatosis by increasing the levels of endocannabinoids, which leads to the activation of cannabinoid receptors.

The concentrations of CPS used in the current study ranged from 0.002 to 20 μM. The low range of concentrations is comparable to previous in vitro studies where the effects of CPS on detoxication systems in hepatocytes were examined (Howell et al., 2016; Rouimi et al., 2012). In addition, these levels are comparable to the peak serum concentrations of CPS (<10 μM) in humans following intentional or accidental exposure to CPS (Eddleston et al., 2005). The current range of CPS concentrations produced marked inhibition of the hepatic serine hydrolases. The sensitivities of the target enzymes in the hepatocytes following CPS exposure followed the rank order Ces>FAAH>MAGL. This order is comparable to those previously reported (Casida and Quistad, 2005; Quistad et al., 2006b). Previous in vivo studies from our lab also demonstrated that hepatic Ces and FAAH were more sensitive to inhibition than hepatic MAGL following acute or subacute (for 10 days) oral exposures of mice to CPS (2 mg/kg) (Howell III et al., 2018; Kondakala et al., 2017). Thus, these results indicate that CPS-mediated inhibition of Ces, FAAH, and possibly MAGL may produce a non-cholinergic toxicity in the liver, in addition to the traditional systemic cholinergic endpoints of OP toxicity.

In conclusion, these in vitro studies have examined the sensitivities of OP targets in the liver using immortalized hepatoma cells and primary hepatocytes. The results indicate that hepatic Ces is the most sensitive of the non-cholinergic endpoints and they are consistent with other in vitro studies. The inhibition of Ces and FAAH following CPS exposure was most pronounced in steatotic MCA cells, whereas MAGL inhibition was less sensitive in steatotic primary hepatocytes compared to normal primary hepatocytes. While the current data indicate both cell type and phenotypic alterations in Ces, FAAH, and MAGL enzymatic activities by CPS/CPO, the underlying mechanisms governing these alterations remain elusive and warrants further investigation. Potential mechanisms such as alterations in enzymatic expression levels as well as the possibility of CPS/CPO sequestration in lipid droplets within steatotic hepatocytes should be explored. The capacity of MCA cells and primary hepatocytes to bioactivate CPS appeared to be decreased after 24 hours in culture, but it did not change from 24 to 48 hours. Thus, the observed effects of CPS on non-cholinergic endpoints of OP toxicity in two different cell culture models under normal and steatotic conditions highlight the effects of cell origin and cellular phenotype on CPS toxicity, as well as reinforces the potential effects of cell culture parameters on OP toxicities in vitro.

Supplementary Material

1

Highlights:

  • Carboxylesterases (Ces) were the most sensitive to chlorpyrifos (CPS) inhibition.

  • Time in culture did not alter CPS bioactivation capacity.

  • Neutral lipid accumulation did not alter cellular bioactivation capacity.

  • Ces in steatotic primary hepatocytes is not as sensitive as in steatotic MCA cells.

6. Acknowledgements:

The current studies were supported in part by a Preliminary Data Grant from the Mississippi State University College of Veterinary Medicine Office of Research and Graduate Studies. Undergraduate research stipend support for L.H. and E.M. was provided by the National Institute of Environmental Health Sciences of the National Institutes of Health under grant# R15ES026791. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Footnotes

Declaration of interests

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.

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