Glucuronidation in the caco-2 human intestinal cell line: Induction of UDP-glucuronosyltransferase 1∗6

doi:10.1016/0006-2952(95)00162-S

Biochemical Pharmacology

Volume 50, Issue 4, 8 August 1995, Pages 557-561

https://doi.org/10.1016/0006-2952(95)00162-S Get rights and content

Abstract

The ability of the differentiated human intestinal cell line, Caco-2, to glucuronidate various endobiotic and xenobiotic molecules was investigated. Glucuronidation of hydroxylated or carboxylic acid compounds such as 1-naphthol, thymol, androsterone, estriol, hyodeoxycholic acid, lithocholic acid, chloramphenicol, paracetamol and morphine could be determined in microsomal fractions of Caco-2 cells. The activity toward 1-naphthol was the highest glucuronidation activity measured in Caco-2 cells. This activity was specifically increased four-fold upon addition of β-naphthoflavone into culture medium but not by rifampicine or clofibrate and was related to a biosynthesis of UDP-glucuronosyltransferase 1^∗6 (UGT1^∗6). α-Naphthoflavone did not affect the inducing property of β-naphthoflavone. 7-Ethoxyresorufin-O-dealkylation activity, supported by cytochrome P4501A1, was induced more than 1000-times in Caco-2 cells by β-naphthoflavone treatment, and this effect was partially abolished by α-naphthoflavone treatment. The results suggest that several isoforms, including UGT1^∗6, are expressed in Caco-2 cells.

References (20)

KW Bock et al.
Paracetamol glucuronidation by recombinant rat and human phenol UDP-glucuronosyltransferases
Biochem Pharmacol
(1993)
M Bradford
A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal Biochem
(1976)
G Otani et al.
UDP-glucuronosyltransferase in perfused rat and in microsomes. III. Effects of galactosamine and carbon tetrachloride on the glucuronidation of 1-naphthol and bilirubin
Biochem Pharmacol
(1976)
MD Burke et al.
Ethoxy-, pentoxy- and benzoxyphenoxazone and homologues: a series of substrates to distinguish between different induced cytochrome P-450
Biochem Pharmacol
(1985)
M Ouzzine et al.
Expression and role of the human liver UDP-glucuronosyltransferase UGT1^∗6 analysed by specific antibodies raised against a hybrid protein produced in Escherichia coli
Arch Biochem Biophys
(1994)
M Daujat et al.
Expression of five forms of microsomal cytochrome P-450 in primary culture of rabbit hepatocytes treated with various classes of inducers
Biochem Pharmacol
(1987)
B Burchell et al.
The UDP glucuronosyltransferase gene superfamily: suggested nomenclature based on evolutionary divergence
DNA Cell Biol
(1991)
GJ Mulder
Glucuronidation and its role in regulation of biological activity of drugs
Ann Rev Pharmacol Toxicol
(1992)
ASj Koster et al.
Glucuronidation in rat intestinal cells. Dependence of glucose supply and resistance to inhibition by ethanol and fasting
Arch Toxicol
(1984)
J Fogh et al.
127 Cultured human colon cell lines producing tumors in nude mice
J Natl Acad Sci USA
(1977)

There are more references available in the full text version of this article.

Cited by (36)

First-pass metabolism via UDP-glucuronosyltransferase: A barrier to oral bioavailability of phenolics
2011, Journal of Pharmaceutical Sciences
Citation Excerpt :
UGT1A1, 1A6, 1A9, and 2B15 contribute significantly to its glucuronidation.147 Glucuronidation of acetaminophen was also observed in Caco-2 cell microsomes.148 However, in Caco-2 transport study, acetaminophen glucuronide was not formed, but apical and basolateral efflux of sulfate was observed.149
Glucuronidation mediated by UDP-glucuronosyltransferases (UGTs) is a significant metabolic pathway that facilitates efficient elimination of numerous endobiotics and xenobiotics, including phenolics. UGT genetic deficiency and polymorphisms or inhibition of glucuronidation by concomitant use of drugs are associated with inherited physiological disorders or drug-induced toxicities. Moreover, extensive glucuronidation can be a barrier to oral bioavailability as the first-pass glucuronidation (or premature clearance by UGTs) of orally administered agents usually results in the poor oral bioavailability and lack of efficacies. This review focused on the first-pass glucuronidation of phenolics including natural polyphenols and pharmaceuticals. The complexity of UGT-mediated metabolism of phenolics is highlighted with species-, gender-, organ- and isoform-dependent specificity, as well as functional compensation between UGT1A and 2B subfamily. In addition, recent advances are discussed with respect to the mechanisms of enzymatic actions, including the important properties such as binding pocket size and phosphorylation requirements. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:3655–3681, 2011
Caco-2 cell monolayers as a tool to study simultaneous phase II metabolism and metabolite efflux of indomethacin, paracetamol and 1-naphthol
2010, International Journal of Pharmaceutics
The human intestinal cell line, Caco-2, was used to study compounds – indomethacin, paracetamol and 1-naphthol – that undergo intestinal phase II metabolism followed by apical and/or basolateral efflux of the metabolites and/or parent compounds. The interplay was studied during permeability experiments across fully differentiated Caco-2 cell monolayers. The parent compounds and their glucuronide and/or sulfate metabolites were detected by LC–MS/MS. Conjugation of the model compounds and effluxes of their metabolites were observed. The efflux of indomethacin glucuronide was apical, but complementary basolateral efflux was observed at the highest indomethacin concentration (500 μM), probably due to apical saturation. Paracetamol glucuronide was not formed in these experiments, but apical and basolateral effluxes of paracetamol sulfate were observed. A typical bell-shaped inhibition curve was observed for the formation of 1-naphthol glucuronides, indicating substrate or product inhibition of the UGT enzyme(s) at higher 1-naphthol concentrations (200 μM and 500 μM). Based on these results, the fully differentiated Caco-2 cell monolayers can be applied as a platform for qualitative in vitro studies, where phase II metabolism and efflux activities are ongoing simultaneously.
Molecular and cellular effects of food contaminants and secondary plant components and their plausible interactions at the intestinal level
2008, Food and Chemical Toxicology
The intestinal mucosa is not simply a barrier allowing entry of compounds such as nutrients or chemicals, and restricting that of others. Intestinal cells and activities perform selective absorption, biotransformations and efflux back to the lumen. Furthermore, food substances affect both bioavailability and intestinal function. Some are able to act as transcriptional regulators and enzyme modulators.
This review points out plausible interactions between food contaminants and/or natural constituents at molecular and cellular levels and focuses on the effects of classical (pesticides and veterinary drugs), environmental (heavy metals, PCBs, dioxins, etc.) and food processing generated (PAHs, heterocyclic amines, etc.) contaminants on absorption, metabolism and efflux. Special attention is given to secondary metabolites of molds (mycotoxins) and plants (polyphenols). Molecular targets are briefly described as well as regulation mechanisms. Where possible, data referred to deal with human intestinal functions in vivo, and with in vitro studies on human intestinal Caco-2 cells; however, since data related to the intestine are rather scarce, effects on molecular targets in liver are also considered.
This review also points out the urgent need for fully validated high throughput in vitro tools to screen combinations of substances, at realistic intestinal concentrations. A higher priority could then be given to combinations of nutrients, xenobiotics and food contaminants, with hazardous or beneficial impacts on human health.
Pyrene-induced CYP1A2 and SULT1A1 may be regulated by CAR and not by AhR
2007, Toxicology
Aryl hydrocarbon receptor (AhR) plays important roles in the regulation and induction of xenobiotic-metabolizing enzymes including the cytochromes P450 1 family (CYP1) and UDP-glucuronosyltransferases 1A (UGT1As) by polycyclic aromatic hydrocarbons as well as chlorinated aromatic hydrocarbons. To determine whether pyrene-induced xenobiotic-metabolizing enzymes are regulated by AhR, male AhR (+/+) and (−/−) mice were used. Both genotyped mice were exposed to 0, 205, 300 or 410 mg/(kg day pyrene), once daily, for four consecutive days by gavage. Exposure to pyrene did not influence hepatic CYP1A1-mRNA in mice of both genotypes, whereas it induced hepatic CYP1A2 protein and mRNA expression and associated 7-ethoxyresorufin O-deethylase and pyrene 1-hydroxylation activities in both AhR (+/+) and (−/−) mice. Similar effects were also found with sulfotransferase 1A1 expression and the associated 1-hydroxypyrene sulfation activity. In contrast, pyrene exposure increased expression of the UGT1A1 and 1A6, and glucuronidation activities associated with 1-hydroxypyrene and 1-naphthol in the liver only in AhR (−/−) mice, although pyrene treatment dose-dependently decreased the latter activity. Pyrene exposure did not increase AhR-mRNA expression in AhR (+/+) mice. In contrast, pyrene-induced expression of the hepatic constitutive androstane receptor (CAR) and one of its target genes, CYP2B10, in both AhR (+/+) and (−/−) mice. These results strongly suggest that pyrene-induced CYP1A2 and SULT1A1 are regulated by CAR, not by AhR. However, the mechanisms of UGT1A1 and 1A6 induction by pyrene were not elucidated in this study.
Essential oils-their antimicrobial activity against Escherichia coli and effect on intestinal cell viability
2006, Toxicology in Vitro
Essential oils are known to possess antimicrobial activity against a wide spectrum of bacteria. The main objective of this study was to evaluate possible harmful effects of four commonly used essential oils and their major components on intestinal cells. Antimicrobial activity of selected plant extracts against enteroinvasive Escherichia coli was dose dependent. However, doses of essential oils with the ability to completely inhibit bacterial growth (0.05%) showed also relatively high cytotoxicity to intestinal-like cells cultured in vitro. Lower doses of essential oils (0.01%) had only partial antimicrobial activity and their damaging effect on Caco-2 cells was only modest. Cell death assessment based on morphological and viability staining followed by fluorescence microscopy showed that essential oils of cinnamon and clove and their major component eugenol had almost no cytotoxic effect at lower doses. Although essential oil of oregano and its component carvacrol slightly increased the incidence of apoptotic cell death, they showed extensive antimicrobial activity even at lower concentrations. Relatively high cytotoxicity was demonstrated by thyme oil, which increased both apoptotic and necrotic cell death incidence. In contrast, its component thymol showed no cytotoxic effect as well as greatly-reduced ability to inhibit visible growth of the chosen pathogen in the doses used. On the other hand, the addition of all essential oils and their components at lower doses, with the exception of thyme oil, to bacterial suspension significantly reduced the cytotoxic effect of E. coli on Caco-2 cells after 1 h culture. In conclusion, it is possible to find appropriate doses of essential oils showing both antimicrobial activity and very low detrimental effect on intestinal cells.
Inducibility of UDP-glucuronosyltransferase 1As by β-naphthoflavone in HepG2 cells
2006, Food and Chemical Toxicology
UDP-glucuronosyltransferases (UGTs) are conjugation enzymes, which are regulated in a tissue-specific manner by endogenous and environmental factors. In this study, we focused on UGT1A isoforms (UGT1A1, UGT1A6 and UGT1A9), mainly expressed in the human liver, and examined the inducibility of UGT1As by β-naphthoflavone (BNF) in human hepatoma HepG2 cells. The cells were pretreated for 72 h with BNF at concentrations of 25, 50 and 100 μM. 7-Ethyl-10-hydroxycamptothecin (SN-38) glucuronidation, used as a probe for UGT1A1, showed sigmoidal kinetics with a Hill coefficient (n) of 1.2–1.3 in control and BNF-pretreated HepG2 cells. The V_max values were significantly increased 3.6- to 4.3-fold by BNF, whereas there was no significant change in the S₅₀ values by BNF at any concentration examined. On the other hand, 4-methylumbelliferone (4-MU) glucuronidation as a probe for UGT1A6 and UGT1A9 in the control and BNF-pretreated HepG2 cells exhibited a biphasic kinetic pattern. Although K_m1 values for the low-K_m phase were similar between the control and BNF-pretreated HepG2 cells, K_m2 values for the high-K_m phase of BNF-pretreated HepG2 cells were reduced to 54–69% of control HepG2 cells. The values of V_max1 and V_max2 for the low- and high-K_m phases, respectively, were significantly increased 1.9- to 2.6-fold by BNF at 25 and/or 50 μM but not 100 μM. With respect to V_max (V_max1 and V_max2) and V_max/K_m (V_max1/K_m1 and V_max2/K_m2), the values were significantly increased 2.0- to 3.2-fold by BNF at all concentrations examined. Furthermore, real-time reverse transcription polymerase chain reaction using TaqMan probes demonstrated that BNF concentration-dependently induced mRNA levels of UGT1A1 but not UGT1A6 or UGT1A9 in HepG2 cells (1.3- to 6.0-fold). These results suggest that the inducibility of UGT1A isoforms in HepG2 cells by BNF is different from other aryl hydrocarbon receptor agonists previously reported, and should provide useful information for the prediction of drug–drug interactions and toxicological assessment of environmental chemicals.

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Biochemical Pharmacology

Abstract

References (20)

Paracetamol glucuronidation by recombinant rat and human phenol UDP-glucuronosyltransferases

Biochem Pharmacol

A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding

Anal Biochem

UDP-glucuronosyltransferase in perfused rat and in microsomes. III. Effects of galactosamine and carbon tetrachloride on the glucuronidation of 1-naphthol and bilirubin

Biochem Pharmacol

Ethoxy-, pentoxy- and benzoxyphenoxazone and homologues: a series of substrates to distinguish between different induced cytochrome P-450

Biochem Pharmacol

Expression and role of the human liver UDP-glucuronosyltransferase UGT1^∗6 analysed by specific antibodies raised against a hybrid protein produced in Escherichia coli

Arch Biochem Biophys

Expression of five forms of microsomal cytochrome P-450 in primary culture of rabbit hepatocytes treated with various classes of inducers

Biochem Pharmacol

The UDP glucuronosyltransferase gene superfamily: suggested nomenclature based on evolutionary divergence

DNA Cell Biol

Glucuronidation and its role in regulation of biological activity of drugs

Ann Rev Pharmacol Toxicol

Glucuronidation in rat intestinal cells. Dependence of glucose supply and resistance to inhibition by ethanol and fasting

Arch Toxicol

127 Cultured human colon cell lines producing tumors in nude mice

J Natl Acad Sci USA

Cited by (36)

First-pass metabolism via UDP-glucuronosyltransferase: A barrier to oral bioavailability of phenolics

Caco-2 cell monolayers as a tool to study simultaneous phase II metabolism and metabolite efflux of indomethacin, paracetamol and 1-naphthol

Molecular and cellular effects of food contaminants and secondary plant components and their plausible interactions at the intestinal level

Pyrene-induced CYP1A2 and SULT1A1 may be regulated by CAR and not by AhR

Essential oils-their antimicrobial activity against Escherichia coli and effect on intestinal cell viability

Inducibility of UDP-glucuronosyltransferase 1As by β-naphthoflavone in HepG2 cells

Short communicationGlucuronidation in the caco-2 human intestinal cell line: Induction of UDP-glucuronosyltransferase 1∗6

Abstract

Biochem Pharmacol

Anal Biochem

Biochem Pharmacol

Biochem Pharmacol

Arch Biochem Biophys

Biochem Pharmacol

The UDP glucuronosyltransferase gene superfamily: suggested nomenclature based on evolutionary divergence

DNA Cell Biol

Glucuronidation and its role in regulation of biological activity of drugs

Ann Rev Pharmacol Toxicol

Glucuronidation in rat intestinal cells. Dependence of glucose supply and resistance to inhibition by ethanol and fasting

Arch Toxicol

127 Cultured human colon cell lines producing tumors in nude mice

J Natl Acad Sci USA

Short communication
Glucuronidation in the caco-2 human intestinal cell line: Induction of UDP-glucuronosyltransferase 1^∗6