Fatty acid ethyl and methyl ester synthases, and fatty acid anilide synthase in HepG2 and AR42J cells: Interrelationships and inhibition by tri- o-tolyl phosphate

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Abstract

Synthesis of fatty acid ethyl esters (FAEEs), fatty acid methyl esters (FAMEs), and fatty acid anilides (FAAs) in humans and/or experimental animals and in vitro have been reported by us and other investigators. In previous studies, we have demonstrated that fatty acid ethyl ester synthase (FAEES), purified from rat liver microsomes, is structurally and functionally identical to the rat liver microsomal carboxylesterase (pI 6.1) and suggested a role in the conjugation of a variety of xenobiotic alcohols with endogenous fatty acids (B. S. Kaphalia, R. R. Fritz, and G. A. S. Ansari, Chem. Res. Toxicol. 11, 211-218, 1997). However, hepatic FAEES was found to be structurally and functionally different from that of pancreas. Therefore, the present study was undertaken to determine structural and functional interrelationships among the enzyme(s) involved in the synthesis of FAEEs, FAMEs, and FAAs, in HepG2 and AR42J cells using tri-o-tolyl phosphate (TOTP), a specific inhibitor for β-esterases. Synthesis of FAEEs, FAMEs, and FAAs, studied in the HepG2 cells, was found to be dose- and time-dependent following incubation with methanol, ethanol, or aniline, respectively. Approximately 86-90% inhibition of FAEE, FAME, and FAA synthesizing activities was found in HepG2 cells following exposure to 2.5 μM TOTP. Identical profiles of dose- and time-dependent inhibition of FAEE, FAME, and FAA synthesizing activities by TOTP in HepG2 cells suggest that synthesis of FAEEs, FAMEs, and FAAs is catalyzed by the same enzyme(s). However, FAEE, FAME, and FAA synthesizing activities in AR42J cells could not be inhibited by TOTP under similar experimental conditions. A differential pattern of p- nitrophenyl acetate hydrolyzing activity (a measure of esterase activity) similar to that of fatty acid ester/anilide synthesizing activities was observed in the two cell lines. These results are further substantiated by the presence of ~60 kDa (subunit molecular weight) protein in the postnuclear fraction of HepG2 but not in AR42J cells by Western blot analysis using antibodies raised against FAEES, purified from rat liver microsomes or adipose tissue. Therefore, the enzyme responsible for the FAEE, FAME, or FAA synthesizing activities is most probably carboxylesterase in HepG2 cells and is structurally and functionally different than that present in AR42J cells. These studies also indicate the utility of HepG2 and AR42J cell cultures as an alternative to the animal model regarding studies on nonoxidative metabolism of alcohols and amines, in general.

Original languageEnglish (US)
Pages (from-to)134-141
Number of pages8
JournalToxicology and Applied Pharmacology
Volume159
Issue number2
DOIs
StatePublished - Sep 1 1999

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Anilides
Fatty Acid Synthases
Hep G2 Cells
Esters
Fatty Acids
tri-o-cresyl phosphate
fatty acyl ethyl ester synthase
Liver
Rats
Carboxylesterase
Liver Microsomes
Esterases

ASJC Scopus subject areas

  • Pharmacology
  • Toxicology

Cite this

@article{7b7dc6581d5d40989e1a7d79929ebb46,
title = "Fatty acid ethyl and methyl ester synthases, and fatty acid anilide synthase in HepG2 and AR42J cells: Interrelationships and inhibition by tri- o-tolyl phosphate",
abstract = "Synthesis of fatty acid ethyl esters (FAEEs), fatty acid methyl esters (FAMEs), and fatty acid anilides (FAAs) in humans and/or experimental animals and in vitro have been reported by us and other investigators. In previous studies, we have demonstrated that fatty acid ethyl ester synthase (FAEES), purified from rat liver microsomes, is structurally and functionally identical to the rat liver microsomal carboxylesterase (pI 6.1) and suggested a role in the conjugation of a variety of xenobiotic alcohols with endogenous fatty acids (B. S. Kaphalia, R. R. Fritz, and G. A. S. Ansari, Chem. Res. Toxicol. 11, 211-218, 1997). However, hepatic FAEES was found to be structurally and functionally different from that of pancreas. Therefore, the present study was undertaken to determine structural and functional interrelationships among the enzyme(s) involved in the synthesis of FAEEs, FAMEs, and FAAs, in HepG2 and AR42J cells using tri-o-tolyl phosphate (TOTP), a specific inhibitor for β-esterases. Synthesis of FAEEs, FAMEs, and FAAs, studied in the HepG2 cells, was found to be dose- and time-dependent following incubation with methanol, ethanol, or aniline, respectively. Approximately 86-90{\%} inhibition of FAEE, FAME, and FAA synthesizing activities was found in HepG2 cells following exposure to 2.5 μM TOTP. Identical profiles of dose- and time-dependent inhibition of FAEE, FAME, and FAA synthesizing activities by TOTP in HepG2 cells suggest that synthesis of FAEEs, FAMEs, and FAAs is catalyzed by the same enzyme(s). However, FAEE, FAME, and FAA synthesizing activities in AR42J cells could not be inhibited by TOTP under similar experimental conditions. A differential pattern of p- nitrophenyl acetate hydrolyzing activity (a measure of esterase activity) similar to that of fatty acid ester/anilide synthesizing activities was observed in the two cell lines. These results are further substantiated by the presence of ~60 kDa (subunit molecular weight) protein in the postnuclear fraction of HepG2 but not in AR42J cells by Western blot analysis using antibodies raised against FAEES, purified from rat liver microsomes or adipose tissue. Therefore, the enzyme responsible for the FAEE, FAME, or FAA synthesizing activities is most probably carboxylesterase in HepG2 cells and is structurally and functionally different than that present in AR42J cells. These studies also indicate the utility of HepG2 and AR42J cell cultures as an alternative to the animal model regarding studies on nonoxidative metabolism of alcohols and amines, in general.",
author = "Bhupendra Kaphalia and Green, {Steven M.} and Ghulam Ansari",
year = "1999",
month = "9",
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journal = "Toxicology and Applied Pharmacology",
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T1 - Fatty acid ethyl and methyl ester synthases, and fatty acid anilide synthase in HepG2 and AR42J cells

T2 - Interrelationships and inhibition by tri- o-tolyl phosphate

AU - Kaphalia, Bhupendra

AU - Green, Steven M.

AU - Ansari, Ghulam

PY - 1999/9/1

Y1 - 1999/9/1

N2 - Synthesis of fatty acid ethyl esters (FAEEs), fatty acid methyl esters (FAMEs), and fatty acid anilides (FAAs) in humans and/or experimental animals and in vitro have been reported by us and other investigators. In previous studies, we have demonstrated that fatty acid ethyl ester synthase (FAEES), purified from rat liver microsomes, is structurally and functionally identical to the rat liver microsomal carboxylesterase (pI 6.1) and suggested a role in the conjugation of a variety of xenobiotic alcohols with endogenous fatty acids (B. S. Kaphalia, R. R. Fritz, and G. A. S. Ansari, Chem. Res. Toxicol. 11, 211-218, 1997). However, hepatic FAEES was found to be structurally and functionally different from that of pancreas. Therefore, the present study was undertaken to determine structural and functional interrelationships among the enzyme(s) involved in the synthesis of FAEEs, FAMEs, and FAAs, in HepG2 and AR42J cells using tri-o-tolyl phosphate (TOTP), a specific inhibitor for β-esterases. Synthesis of FAEEs, FAMEs, and FAAs, studied in the HepG2 cells, was found to be dose- and time-dependent following incubation with methanol, ethanol, or aniline, respectively. Approximately 86-90% inhibition of FAEE, FAME, and FAA synthesizing activities was found in HepG2 cells following exposure to 2.5 μM TOTP. Identical profiles of dose- and time-dependent inhibition of FAEE, FAME, and FAA synthesizing activities by TOTP in HepG2 cells suggest that synthesis of FAEEs, FAMEs, and FAAs is catalyzed by the same enzyme(s). However, FAEE, FAME, and FAA synthesizing activities in AR42J cells could not be inhibited by TOTP under similar experimental conditions. A differential pattern of p- nitrophenyl acetate hydrolyzing activity (a measure of esterase activity) similar to that of fatty acid ester/anilide synthesizing activities was observed in the two cell lines. These results are further substantiated by the presence of ~60 kDa (subunit molecular weight) protein in the postnuclear fraction of HepG2 but not in AR42J cells by Western blot analysis using antibodies raised against FAEES, purified from rat liver microsomes or adipose tissue. Therefore, the enzyme responsible for the FAEE, FAME, or FAA synthesizing activities is most probably carboxylesterase in HepG2 cells and is structurally and functionally different than that present in AR42J cells. These studies also indicate the utility of HepG2 and AR42J cell cultures as an alternative to the animal model regarding studies on nonoxidative metabolism of alcohols and amines, in general.

AB - Synthesis of fatty acid ethyl esters (FAEEs), fatty acid methyl esters (FAMEs), and fatty acid anilides (FAAs) in humans and/or experimental animals and in vitro have been reported by us and other investigators. In previous studies, we have demonstrated that fatty acid ethyl ester synthase (FAEES), purified from rat liver microsomes, is structurally and functionally identical to the rat liver microsomal carboxylesterase (pI 6.1) and suggested a role in the conjugation of a variety of xenobiotic alcohols with endogenous fatty acids (B. S. Kaphalia, R. R. Fritz, and G. A. S. Ansari, Chem. Res. Toxicol. 11, 211-218, 1997). However, hepatic FAEES was found to be structurally and functionally different from that of pancreas. Therefore, the present study was undertaken to determine structural and functional interrelationships among the enzyme(s) involved in the synthesis of FAEEs, FAMEs, and FAAs, in HepG2 and AR42J cells using tri-o-tolyl phosphate (TOTP), a specific inhibitor for β-esterases. Synthesis of FAEEs, FAMEs, and FAAs, studied in the HepG2 cells, was found to be dose- and time-dependent following incubation with methanol, ethanol, or aniline, respectively. Approximately 86-90% inhibition of FAEE, FAME, and FAA synthesizing activities was found in HepG2 cells following exposure to 2.5 μM TOTP. Identical profiles of dose- and time-dependent inhibition of FAEE, FAME, and FAA synthesizing activities by TOTP in HepG2 cells suggest that synthesis of FAEEs, FAMEs, and FAAs is catalyzed by the same enzyme(s). However, FAEE, FAME, and FAA synthesizing activities in AR42J cells could not be inhibited by TOTP under similar experimental conditions. A differential pattern of p- nitrophenyl acetate hydrolyzing activity (a measure of esterase activity) similar to that of fatty acid ester/anilide synthesizing activities was observed in the two cell lines. These results are further substantiated by the presence of ~60 kDa (subunit molecular weight) protein in the postnuclear fraction of HepG2 but not in AR42J cells by Western blot analysis using antibodies raised against FAEES, purified from rat liver microsomes or adipose tissue. Therefore, the enzyme responsible for the FAEE, FAME, or FAA synthesizing activities is most probably carboxylesterase in HepG2 cells and is structurally and functionally different than that present in AR42J cells. These studies also indicate the utility of HepG2 and AR42J cell cultures as an alternative to the animal model regarding studies on nonoxidative metabolism of alcohols and amines, in general.

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