Carboxylesterase Inhibitor Bis Research Articles

Development of an in vitro screening method of acute cytotoxicity of the pyrrolizidine alkaloid lasiocarpine in human and rodent hepatic cell lines by increasing susceptibility

Ethnopharmacological relevancePyrrolizidine alkaloids (PAs) are secondary plant ingredients formed in many plant species to protect against predators. PAs are generally considered acutely hepatotoxic, genotoxic and carcinogenic. Up to now, only few in vitro and in vivo investigations were performed to evaluate their relative toxic potential. Aim of the studyThe aim was to develop an in vitro screening method of their cytotoxicity. Materials and methodsHuman and rodent hepatocyte cell lines (HepG2 and H-4-II-E) were used to assess cytotoxicity of the PA lasiocarpine. At concentrations of 25 µM up to even 2400 µM, no toxic effects in neither cell line was observed with standard cell culture media. Therefore, different approaches were investigated to enhance the susceptibility of cells to PA toxicity (using high-glucose or galactose-based media, induction of toxifying cytochromes, inhibition of metabolic carboxylesterases, and inhibition of glutathione-mediated detoxification). ResultsGalactose-based culture medium (11.1 mM) increased cell susceptibility in both cell-lines. Cytochrome P450-induction by rifampicin showed no effect. Inhibition of carboxylesterase-mediated PA detoxification by specific carboxylesterase 2 inhibitor loperamide (2.5 µM) enhanced lasiocarpine toxicity, whereas the unspecific carboxylesterase inhibitor bis(4-nitrophenyl)phosphate (BNPP, 100 µM)) had a weaker effect. Finally, the inhibition of glutathione-mediated detoxification by buthionine sulphoximine (BSO, 100 µM) strongly enhanced lasiocarpine toxicity in H-4-II-E cells in low and medium, but not in high concentrations. ConclusionsIf no toxicity is observed under standard conditions, susceptibility enhancement by using galactose-based media, loperamide, and BSO may be useful to assess relative acute cytotoxicity of PAs in different cell lines.

Pharmacological effects of a novel isosorbide-based butyrylcholinesterase inhibitor

Isosorbide-2-benzylcarbamate-5-benzoate, a novel butyrylcholinesterase inhibitor, shows interspecies variation in its inhibitory activity (IC 50 of 4.3 nM for human plasma butyrylcholinesterase, but 1.09 μM for mouse plasma butyrylcholinesterase). Stability studies revealed that this drug is resistant to hydrolysis by human plasma (no degradation in 1 h). However, it was found to undergo rapid degradation when incubated with mouse plasma or mouse liver homogenate, yielding benzyl carbamate and benzoic acid. The addition of the carboxylesterase inhibitor bis-(4-nitrophenyl) phosphate (BNPP) inhibited the degradation of the novel drug, indicating that it may be a substrate for both butyrylcholinesterase and carboxylesterase. The absence of carboxylesterase from human plasma explains the drug's stability in this medium. In vivo, pharmacodynamic studies on single doses of 1 mg/kg to naïve male C57BL/6 mice revealed maximal plasma butyrylcholinesterase inhibition 20 min after intraperitoneal administration (∼60% inhibition) and 1 h after administration by gavage (∼45% inhibition). While this plasma butyrylcholinesterase inhibition was short-lived, the drug also penetrated the blood–brain barrier resulting in a slight (10–15%) but persistent (≥72 h) reduction in brain butyrylcholinesterase activity.

Vinyl acetate induces intracellular acidification in mouse oral buccal epithelial cells

Vinyl acetate exposure in drinking water has been associated with tumor formation in the upper gastrointestinal tract of rats and mice. One potential mechanism for inducing carcinogenesis involves acidification of the intracellular environment due to the metabolism of vinyl acetate to acetic acid. Prolonged intracellular acidification is thought to produce cytotoxic and/or mitogenic responses that are the sentinel pharmacodynamic steps toward cancer. To determine whether exposure to vinyl acetate affects the intracellular pH of intact oral cavity tissue, isolated mouse oral buccal epithelium was loaded with the pH-sensitive dye BCECF, and then exposed to vinyl acetate concentrations ranging from 10 to 1000 μM for up to 4 min. Extracellular vinyl acetate exposure induced a progressive intracellular acidification that was reversible upon removal of the vinyl acetate. The rate of the acidification was concentration-dependent and increased exponentially within the concentration range tested. The magnitude of the vinyl acetate-induced acidification was inhibited by pretreatment with the carboxylesterase inhibitor bis( p-nitrophenyl)phosphate. These results are consistent with the hypothesis that vinyl acetate contributes to the generation and progression of oral cavity tumors via a process of intracellular acidification. Such a process has been proposed to have practical dose–response thresholds below which the intracellular environment can be maintained within homeostatic bounds and the contribution of exposure to carcinogenic risk is negligible.

Vinyl acetate-induced intracellular acidification: implications for risk assessment.

Cancerbioassays have demonstrated the carcinogenic activity of vinyl acetate in rodents. Tumors appear only at the site of contact and mechanistic data suggest that the carcinogenic mechanism involves carboxylesterase-mediated metabolism of vinyl acetate to acetic acid. It has been hypothesized that intracellular formation of acetate causes a reduction of intracellular pH (pH(i)) at noncytotoxic levels, but that prolonged exposure to reduced pH(i) is cytotoxic and/or mitogenic and drives proliferative responses. Coupled with exposure to metabolically formed acetaldehyde at high administered concentrations, nonlinear dose-response curves for epithelial tumors are produced. Freshly isolated rat hepatocytes were used as a model system to test the concept that exposure of cells to vinyl acetate causes a reduction in pH(i). Quantitative fluorescence imaging ratio microscopy showed that exposure of hepatocytes to vinyl acetate concentrations ranging from 10 to 1000 microM caused rapid and sustained reductions of approximately 0.03 to 0.65 pH units. Cellular acidification was rapidly reversed to control pH(i) upon removal of vinyl acetate. There was minimal accumulation of protons during the exposure period, as suggested by minor differences in pH(i) of cells with or without prior exposure to vinyl acetate. The effect of vinyl acetate on pH(i) was attenuated by prior exposure to the carboxylesterase inhibitor bis(p-nitrophenyl)phosphate. These results support the concept that intracellular acidification is a sentinel pharmacodynamic response of cells to vinyl acetate exposure and that pH(i) is an appropriate metric dose for use in quantitative risk assessments of cancer and noncancer human health risk assessment.

SENSORY NERVE-MEDIATED NASAL VASODILATORY RESPONSE TO INSPIRED ETHYL ACRYLATE

The irritants acrolein, acetaldehyde, and acetic acid induce a rapid sensory nerve-mediated nasal vasodilatory response in the rat. The aim of the current study was to examine acute nasal sensory nerve-mediated acute responses to an irritant ester vapor, ethyl acrylate. For this purpose, the upper respiratory tract of the urethane-anesthetized male F344 rat was isolated by insertion of an endotracheal cannula, and ethyl acrylate-laden air was drawn continuously through that site at a flow rate of 100 ml/min for 50 min. Vascular function was monitored by measuring inert vapor (acetone) uptake throughout the exposure. Nasal flow resistance was also monitored during exposure, and plasma protein extravasation was measured by Evans blue dye leakage. At exposure concentrations of 100 to 400 ppm, ethyl acrylate induced a rapid nasal vasodilatory response, as indicated by increased acetone uptake rates. This response was maintained throughout the exposure. Changes in nasal flow resistance or in Evans blue dye leakage were not observed at these exposure concentrations. The vasodilatory response was diminished in animals pretreated with the sensory nerve toxin capsaicin, providing strong evidence that this response was sensory nerve mediated. Pretreatment with the carboxylesterase inhibitor bis- para -nitro-phenolphospahte at a dose sufficient to inhibit nasal carboxylesterase did not alter the response, suggesting that the parent ester, not the carboxylesterase metabolites, is primarily responsible for the sensory-nerve-mediated vasodilatory responses to this ester.

Effect of the carboxylesterase inhibitor bis-(4-nitrophenyl)phosphate in vivo on aspirin hydrolase and carboxylesterase activities at first-pass sites of metabolism in the guinea pig

Effect of the carboxylesterase inhibitor bis-(4-nitrophenyl)phosphate in vivo on aspirin hydrolase and carboxylesterase activities at first-pass sites of metabolism in the guinea pig

Partial purification and characterization of a microsomal carboxylesterase specific for salicylate esters from guinea-pig liver

Studies on liver carboxylesterases have predominantly involved the use of uncharged ester and amide substrates to monitor activity. A microsomal carboxylesterase (EC 3.1.1.1) from guinea-pig liver microsomes has been identified which specifically hydrolyses aspirin (White, K.N. and Hope, D.B. (1981) Biochem. J. 197, 771–773), a substrate which is negatively charged at physiological pH, and this work describes its partial purification and characterization. The enzyme is monomeric, it has a molecular weight of approx. 55 000 and is very sensitive to inhibition by the carboxylesterase inhibitor bis(4-nitrophenyl)phosphate. Although it could not be completely separated from contaminating carboxylesterases, substrate specificity was investigated using the negatively charged esters of salicylic acid. The enzyme is not specific for the acetyl ester of salicyclic acid, aspirin, but hydrolyses the longer chain esters more rapidly, with the highes V ax for the n-octanoyl ester. The enzyme was subject to substrate inhibition which increased with increasing chain length of the fatty acid on the ester, and approached 100% inhibition at concentrations of substrate below critical micellar concentrations.

Identification of aspirinase with one of the carboxylesterases requiring a thiol group.

Aspirin-hydrolysing activity in guinea-pig liver is located mainly in the microsomal fraction. This activity was found by electrophoresis to be due to a single carboxylesterase band, out of 12 bands revealed with alpha-naphthyl acetate as substrate. The activity is inhibited completely and irreversibly by the carboxylesterase inhibitor bis-(-4-nitrophenyl) hydrogen phosphate, and also by thiol-blocking reagents.

Inhibition of sulphation of phenolic substances by the carboxylesterase inhibitor bis-( p-nitrophenyl)-phosphate in the rat in vivo

Inhibition of sulphation of phenolic substances by the carboxylesterase inhibitor bis-( p-nitrophenyl)-phosphate in the rat in vivo

Effects of the carboxylesterase inhibitor bis-( p-nitrophenyl)-phosphate on elimination of hexobendine

A study was conducted of the effect of pretreatment of rats with the carboxylesterase inhibitor, bis-( p-nitrophenyl)-phosphate (BNPP) on the biliary and urinary excretion of intravenously-administered 14C-hexobendine (HB) and its metabolites. Inhibition of HB ester cleavage by BNPP produced a marked decrease in the urinary excretion of HB hydrolysis products: this effect was accompanied by a definite increase in the biliary excretion of conjugates of O-demethylated HB metabolites. The results indicate the participation of complementary metabolic and excretory pathways in the elimination of HB; this hypothesis is further supported by the finding that BNPP did not alter the half-life of the plasma 14C level.

Effects of the carboxylesterase inhibitor bis-( p-nitrophenyl)-phosphate on disposition and metabolism of hexobendine

The effect of pretreatment with the carboxylesterase inhibitor bis-( p-nitrophenyl)-phosphate (BNPP) on the disposition and metabolism of intravenously-administered [ 14C]hexobendine was studied in rats. Inhibition of hexobendine ester cleavage by BNPP produced a marked diminution in the concentrations of the hexobendine hydrolysis products, 3,4,5-trimethoxybenzoic acid and N,N′-dimethyl-N-[3-(3′,4′,5′-trimethoxybenzoxy)-propyl]-N′-3-hydroxypropyl-ethylendiamine in plasma and in the liver and kidneys. A simultaneous elevation occurred in the levels of demethylated metabolites following BNPP pretreatment. The concentration-time relationship of non-metabolized [ 14C]hexobendine was not significantly altered by BNPP pretreatment. The results suggest that BNPP pretreatment produced a shift towards the dealkylating pathway in hexobendine metabolism. Consequently, esterase inhibition did not significantly affect the hexobendine plasma and tissue levels.

Drug-induced porphyrin biosynthesis. X. Potentiation of propanidid-induced elevation of delta-aminolevulinic acid synthetase and porphyrins in chick embryo liver by the carboxylesterase inhibitor bis-(p-nitrophenyl) phosphate.

Propanidid, an ultra short-acting non-barbiturate anesthetic containing an ester group, induces δ-aminolevulinic acid (ALA)-synthetase and porphyrin accumulation in 17-day-old chick embryo liver. The potency and duration of action of propanidid in inducing ALA-synthetase activity and porphyrin accumulation was markedly increased when administered to chick embryos which had been pretreated with bis-[p-nitrophenyl] phosphate, an inhibitor of liver carboxylesterase.

Inhibition of phenacetin- and acetanilide-induced methemoglobinemia in the rat by the carboxylesterase inhibitor Bis-[ p-nitrophenyl] phosphate

The formation of methemoglobin caused by phenacetin and acetanilide in rats is inhibited by simultaneous treatment with if Bis-[ p-nitrophenyl] phosphate (BNPP), a new carboxylesterase inhibitor, while the methemoglobinemia after p-phenetidine is not influenced by BNPP. The duration of BNPP action on phenacetin-induced methemoglobinemia and the spontaneous return of liver esterase activity after a single dose of BNPP have been studied. The enzymatic hydrolysis of the amide bond of phenacetin by rat liver, in vivo and in vitro, is inhibited considerably less by BNPP than the hydrolysis of acetanilide, monoethylglycine-2,6-xylidide and methyl butyrate, and another amidase may be involved in the deacetylation of phenacetin by rat liver.