Regular ArticlePurification and Characterization of Two Rat-Liver Microsomal Carboxylesterases (Hydrolase A and B)
Abstract
The enzymatic hydrolysis of para-nitrophenylacetate by rat liver microsomes is predominantly catalyzed by two esterases: one with high affinity (Km ∼25 μM) and one with low affinity (Km ∼400 μM) for the substrate. Two kinetically distinct esterases were similarly detected in liver microsomes from mouse, hamster, guinea pig, rabbit, cat, cynomolgus monkey, and human, but only the high-affinity enzyme was detectable in dog liver microsomes. The tissue distribution of these kinetically distinct esterases was examined in rats. High-affinity (Km 20-35 μM esterase activity toward para-nitrophenylacetate was detected in testis, lung, prostate, and pancreas. The activity in testicular microsomes was comparable to that in liver microsomes. Low-affinity (Km 200-700 μM) esterase activity was detected in kidney, small intestine, lung, spleen, heart, and brain. The activity in kidney microsomes was comparable to that in liver microsomes. The high-affinity esterase in testicular and liver microsomes was highly sensitive to the inhibitory effects of phenylmethylsulfonyl fluoride (PMSF), whereas the low-affinity esterase in kidney and liver microsomes was relatively resistant. These results suggested that rat liver microsomes contain two esterases with high activity toward para-nitrophenylacetate, a PMSF-sensitive esterase with high substrate affinity, and a PMSF-insensitive esterase with low substrate affinity. In support of this hypothesis, we have purified and characterized two esterases, designated hydrolases A and B, which appear to be the only abundant enzymes in rat liver microsomes that rapidly hydrolyze para-nitrophenylacetate. Hydrolase A hydrolyzed para-nitrophenylacetate with high affinity (Km ∼25 μM), and was inhibited by extremely low concentrations of PMSF (IC50 ∼100 nM). In contrast, hydrolase B hydrolyzed para-nitrophenylacetate with low affinity (Km ∼400 μM) and was inhibited only by relatively high concentrations of PMSF (IC50 ∼100 μM). Paraoxon, the active metabolite of parathion, and cresylbenzodioxaphosphorin oxide, the active metabolite of tri-ortho-tolylphosphate, completely inhibited the hydrolysis of para-nitrophenylacetate by rat liver microsomes and by hydrolases A and B, whereas the sulfhydryl agent, para-chloromercurobenzoate, was not inhibitory. These results suggest that hydrolases A and B are both serine esterases. The N-terminal amino acid sequences of hydrolases A and B were similar but distinct (23 of the first 30 amino acid residues were identical), indicating that these two esterases are isozymes. Hydrolases A and B bound avidly to concanavalin A, indicating both proteins are glycosylated. Enzymatic removal of the carbohydrate residues with α-mannosidase or endoglycosidase did not decrease the enzymatic activity of hydrolase a or B, although deglycosylation caused hydrolase A to adsorb strongly to glass and plastic. In addition to differences in their substrate affinity and sensitivity to PMSF, hydrolase A (57 kDa, pI ∼5.7) and hydrolase B (59 kDa, pI ∼6.5) could be distinguished by differences in their molecular weight and isoelectric point, and by differences in their substrate specificity. Hydrolase B, but not hydrolase A, had high amidase activity toward acetanilide, whereas hydrolase A but not hydrolase B catalyzed the transesterification of cocaine to ethylcocaine in the presence of ethanol. By comparing the Vmax values of the purified and membrane-bound esterases, hydrolases A and B were estimated to comprise 1.5 and 0.5% of the microsomal protein, respectively. These results suggest that hydrolases A and B correspond respectively to the high- and low-affinity carboxylesterases in rat liver microsomes, and that high levels of hydrolase A are also present in testicular microsomes, whereas high levels of hydrolase B are present in kidney microsomes.
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Effects of chlorpyrifos on non-cholinergic toxicity endpoints in immortalized and primary rat hepatocytes under normal and hepatosteatotic conditions
2022, Toxicology in VitroChlorpyrifos (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.
Biochemical metal accumulation effects and metalloprotein metal detoxification in environmentally exposed tropical Perna perna mussels
2021, Ecotoxicology and Environmental SafetyMarine bivalves have been widely applied as environmental contamination bioindicators, although studies concerning tropical species are less available compared to temperate climate species. Assessments regarding Perna perna mytilid mussels, in particular, are scarce, even though this is an extremely important species in economic terms in tropical countries, such as Brazil. To this end, Perna perna mytilids were sampled from two tropical bays in Southeastern Brazil, one anthropogenically impacted and one previously considered a reference site for metal contamination. Gill metallothionein (MT), reduced glutathione (GSH), carboxylesterase (CarbE) and lipid peroxidation (LPO) were determined by UV−vis spectrophotometry, and metal and metalloid contents were determined by inductively coupled plasma mass spectrometry (ICP−MS). Metalloprotein metal detoxification routes in heat-stable cellular gill fractions were assessed by size exclusion high performance chromatography (SEC−HPLC) coupled to an ICP−MS. Several associations between metals and oxidative stress endpoints were observed at all four sampling sites through a Principal Component Analysis. As, Cd, Ni and Se contents, in particular, seem to directly affect CarbE activity. MT is implicated in playing a dual role in both metal detoxification and radical oxygen species scavenging. Differential SEC−HPLC−ICP−MS metal-binding profiles, and, thus, detoxification mechanisms, were observed, with probable As-, Cu- and Ni-GSH complexation and binding to low molecular weight proteins. Perna perna mussels were proven adequate tropical bioindicators, and further monitoring efforts are recommended, due to lack of data regarding biochemical metal effects in tropical species. Integrated assessments, as performed herein demonstrate, are invaluable in evaluating contaminated aquatic environments, resulting in more accurate ecological risk assessments.
Inhibition of cholinergic and non-cholinergic targets following subacute exposure to chlorpyrifos in normal and high fat fed male C57BL/6J mice
2018, Food and Chemical ToxicologyThe effects of obesity on organophosphate pesticide-mediated toxicities, including both cholinergic and non-cholinergic targets, have not been fully elucidated. Therefore, the present study was designed to determine if high fat diet intake alters the effects of repeated exposure to chlorpyrifos (CPS) on the activities of both cholinergic and noncholinergic serine hydrolase targets. Male C57BL/6J mice were placed on either standard rodent chow or high fat diet for four weeks with CPS exposure (2.0 mg/kg) for the last 10 days of diet intake. Exposure to CPS did not alter acetylcholinesterase in the central nervous system, but it did significantly inhibit circulating cholinesterase activities in both diet groups. CPS significantly inhibited hepatic carboxylesterase and fatty acid amide hydrolase and this inhibition was significantly greater in high fat fed animals. Additionally, CPS exposure and high fat diet intake downregulated genes involved in hepatic de novo lipogenesis as well as cytochrome P450 enzymes involved in hepatic xenobiotic metabolism. In summary, the present study demonstrates that high fat diet intake potentiates CPS mediated inhibition of both carboxylesterase and fatty acid amide hydrolase in the liver of obese animals following subacute exposure and suggests obesity may be a risk factor for increased non-cholinergic hepatic CPS toxicity.
Effects of high-fat diet and age on the blood lipidome and circulating endocannabinoids of female C57BL/6 mice
2018, Biochimica et Biophysica Acta - Molecular and Cell Biology of LipidsAlterations in lipid metabolism play a significant role in the pathogenesis of obesity-associated disorders, and dysregulation of the lipidome across multiple diseases has prompted research to identify novel lipids indicative of disease progression. To address the significant gap in knowledge regarding the effect of age and diet on the blood lipidome, we used shotgun lipidomics with electrospray ionization-mass spectrometry (ESI-MS). We analyzed blood lipid profiles of female C57BL/6 mice following high-fat diet (HFD) and low-fat diet (LFD) consumption for short (6 weeks), long (22 weeks), and prolonged (36 weeks) periods. We examined endocannabinoid levels, plasma esterase activity, liver homeostasis, and indices of glucose tolerance and insulin sensitivity to compare lipid alterations with metabolic dysregulation. Multivariate analysis indicated differences in dietary blood lipid profiles with the most notable differences after 6 weeks along with robust alterations due to age. HFD altered phospholipids, fatty acyls, and glycerolipids. Endocannabinoid levels were affected in an age-dependent manner, while HFD increased plasma esterase activity at all time points, with the most pronounced effect at 6 weeks. HFD-consumption also altered liver mRNA levels of PPARα, PPARγ, and CD36. These findings indicate an interaction between dietary fat consumption and aging with widespread effects on the lipidome, which may provide a basis for identification of female-specific obesity- and age-related lipid biomarkers.
Effects of acute exposure to chlorpyrifos on cholinergic and non-cholinergic targets in normal and high-fat fed male C57BL/6J mice
2017, Toxicology and Applied PharmacologyThe prevalence of obesity is increasing at an alarming rate in the United States with 36.5% of adults being classified as obese. Compared to normal individuals, obese individuals have noted pathophysiological alterations which may alter the toxicokinetics of xenobiotics and therefore alter their toxicities. However, the effects of obesity on the toxicity of many widely utilized pesticides has not been established. Therefore, the present study was designed to determine if the obese phenotype altered the toxicity of the most widely used organophosphate (OP) insecticide, chlorpyrifos (CPS). Male C57BL/6J mice were fed normal or high-fat diet for 4 weeks and administered a single dose of vehicle or CPS (2.0 mg/kg; oral gavage) to assess cholinergic (acetylcholinesterase activities) and non-cholinergic (carboxylesterase and endocannabinoid hydrolysis) endpoints. Exposure to CPS significantly decreased red blood cell acetylcholinesterase (AChE) activity, but not brain AChE activity, in both diet groups. Further, CPS exposure decreased hepatic carboxylesterase activity and hepatic hydrolysis of a major endocannabinoid, anandamide, in a diet-dependent manner with high-fat diet fed animals being more sensitive to CPS-mediated inhibition. These in vivo studies were corroborated by in vitro studies using rat primary hepatocytes, which demonstrated that fatty acid amide hydrolase and CES activities were more sensitive to CPS-mediated inhibition than 2-arachidonoylglycerol hydrolase activity. These data demonstrate hepatic CES and FAAH activities in high-fat diet fed mice were more potently inhibited than those in normal diet fed mice following CPS exposure, which suggests that the obese phenotype may exacerbate some of the non-cholinergic effects of CPS exposure.
Alteration of syncytiotrophoblast mitochondria function and endothelial nitric oxide synthase expression in the placenta of rural residents
2016, Reproductive ToxicologyThe impact of environmental organophosphate (OP) pesticide exposure on respiratory complexes, enzymatic antioxidant defense activities, and oxidative damage markers in the syncytiotrophoblast and cytotrophoblast mitochondria was evaluated. Placental progesterone (PG) levels and endothelial nitric oxide synthase (eNOS) expression were studied. Samples from women non-exposed (control group-CG) and women living in a rural area (rural group-RG) were collected during pesticide spraying season (RG-SS) and non-spraying season (RG-NSS).
In RG-SS, the exposure biomarker placental carboxylesterase decreased and syncytiotrophoblast cytochrome c oxidase activity increased, while 4-hydroxynonenal levels decreased. PG levels decreased in RG-SS and in the RG. Nitric oxide synthase expression decreased in RG, RG-SS and RG-NSS. No significant changes in mitochondrial antioxidant enzyme activities were found. These results suggest that the alteration of syncytiotrophoblast mitochondrial complex IV activity and steroidogenic function may be associated to pesticide exposure. Reduction in placental PG and eNOS expression may account for low newborn weight in RG.