Phenobarbital inducible UDP-glucuronosyltransferase is responsible for glucuronidation of 3′-azido-3′-deoxythymidine: Characterization of the enzyme in human and rat liver microsomes

doi:10.1016/0003-9861(90)90442-2

Archives of Biochemistry and Biophysics

Volume 281, Issue 2, September 1990, Pages 264-270

https://doi.org/10.1016/0003-9861(90)90442-2 Get rights and content

Abstract

Glucuronidation by liver microsomes of 3′-azido-3′-deoxythymidine (AZT) was characterized in human and in various animal species. The glucuronide isolated by HPLC, was identified by mass spectrometry (fast atom bombardment, desorption in chemical ionization), and β-glucuronidase hydrolysis. AZT glucuronidation reaction in liver microsomes of human and monkey proceeded similarly with an apparent V_max of 0.98 nmol/min/mg protein and apparent K_m of 13 mm. Oleoyl lysophosphatidylcholine activated more than twofold the formation of the glucuronide. Human kidney microsomes could also biosynthesize AZT glucuronide, although to a lower extent (six times less than the corresponding liver). Probenecid, which is administered to AIDS patients, decreased hepatic AZT glucuronidation in vitro (I₅₀ =1.5 mm), whereas paracetamol did not exert any effect at concentrations up to 21.5 mm. Morphine also inhibited the reaction (I₅₀ = 2.7 mm). AZT glucuronidation presented the highest rate in human and in monkey (0.50 nmol/min/mg protein); pig and rat glucuronidated the drug two and three times less, respectively. In Gunn rat, the specific activity in liver microsomes was similar (0.18 nmol/min/mg protein) to that of the congenic normal strain; this suggests that an isozyme other than bilirubin UDP-glucuronosyltransferase catalyzed the reaction. In rats, AZT glucuronidation was stimulated fourfold by phenobarbital; 3-methylcholanthrene or clofibrate failed to increase this activity. This result was consistent with the bulkiness of the AZT molecule (thickness 6.7 Å), which is a critical structural factor for glucuronidation of the drug by phenobarbital-induced isozymes. Altogether, the results strongly indicate that UDP-glucuronosyltransferase (phenobarbital inducible forms) is responsible for AZT glucuronidation.

References (28)

G. Siest et al.
Biochem Pharmacol
(1987)
J. Thomassin et al.
Comp. Biochem. Physiol
(1986)
G.H. Hogeboom
M.M. Bradford
Anal. Biochem
(1976)
A. Colin-Neiger et al.
J. Biochem. Biophys. Methods
(1984)
J. Magdalou et al.
J. Biol. Chem
(1982)
I. Okulicz-Kozaryn et al.
Biochem. Pharmacol
(1981)
A. Resetar et al.
Biochem. Pharmacol
(1989)
S. Dragacci et al.
Biochem. Pharmacol
(1987)
A. Cartier et al.
Chemomet. Intel. Lab. Sys
(1987)

J. Thomassin et al.

Eur. J. Med. Chem

(1987)

J.A. Boutin et al.

Comp. Biochem. Physiol

(1987)

A. Certain et al.

J. Pharmac. Clin

(1989)

S.S. Good et al.

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Citation Excerpt :
Two Km values have been reported for this reaction and are very different, one is reported as 1600 μM and the other is substantially lower at approximately 100 μM [10,11]. UGT2B7 is the only UGT isoform reported to glucuronidate AZT and the Km for human liver microsomal glucuronidation of AZT has been estimated at between 2.2 and 4.2 mM [27–32] and in one instance reported to be as high as 13 mM [33] although this value does seem extremely high. Competitive inhibition simulations were run using the high and low Km values reported and the results are shown in Fig. 5A and B.
Valproic acid glucuronidation kinetics were carried out with three human UGT isoforms: UGT1A6, UGT1A9, and UGT2B7 as well as human liver and kidney microsomes. The glucuronidation of valproic acid was typified by high K_m values with microsomes and expressed UGTs (2.3–5.2 mM). The ability of valproic acid to interact with the glucuronidation of drugs, steroids and xenobiotics in vitro was investigated using the three UGT isoforms known to glucuronidate valproic acid. In addition to this the effect of valproic acid was investigated using two other UGT isoforms: UGT1A1 and UGT2B15 which do not glucuronidate valproic acid. Valproic acid inhibited UGT1A9 catalyzed propofol glucuronidation in an uncompetitive manner and UGT2B7 catalyzed AZT glucuronidation competitively (K_i=1.6±0.06 mM). Valproate significantly inhibited UGT2B15 catalyzed steroid and xenobiotic glucuronidation although valproate was not a substrate for this UGT isoform. No significant inhibition of UGT1A1 or UGT1A6 by valproic acid was observed. These data indicate that valproic acid inhibition of glucuronidation reactions is not always due to simple competitive inhibition of substrates.
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Glucuronidation is a major detoxification pathway in vertebrates. The reaction is catalyzed by a family of UDP-glucuronosyltransferases (UGTs) and involves conjugation of many endobiotic and xenobiotic substances with glucuronic acid, forming inactive water-soluble glucuronides. UGT prevents the accumulation of potentially toxic compounds and/or their subsequent bioactivation to more toxic intermediates, although biologically active glucuronides are also known. Impairment of UGTs may have important toxicological consequences. Substances found to inhibit or down-regulate UGT activity include endogenous compounds, a wide range of clinically used drugs, environmental contaminants, and natural toxic substances present in the diet. The development of selective, active-site-directed UGT inhibitors greatly enables the study of various UGT isoenzymes. A promising approach offers the design of transition-state analogs of the glucuronidation reaction.
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1998, Journal of Chromatography A
An immobilized HPLC column has been developed for the on-line deconjugation of β-glucuronides. The enzymatic activity of this column has been previously demonstrated [1]. This study reports on the application of the immobilized β-glucuronidase column to the analysis of glucuronide metabolites in the urine. The system utilized in this work was composed of an internal-surface reversed-phase (ISRP) column (50×4.6 mm) containing a hydrophobic inner phase and a hydrophilic outer phase, a β-glucuronidase immobilized enzyme reactor (BG-IMER) column (50×4.6 mm) and a C₈ reversed-phase column (150×4.6 mm). The columns were connected with three six-port switching valves. A coupled-column procedure was developed for urine samples containing chloramphenicol-β-d-glucuronides (0.07–1.1 mM/injection). Urine samples were injected into the ISRP column where the glucuronides were separated from the biological matrix, with matrix contaminants eluting off-line to waste. Eluent from the ISRP column containing the glucuronides was then transferred on-line to the β-glucuronidase column for deconjugation and passed directly on-line to the C₈ column. In this portion of the chromatographic procedure, the mobile phase consisted of 0.01 M ammonium acetate at pH 6.7. The analyte concentrated on the top of the reversed-phase column was then eluted using a gradient mobile phase system of acetonitrile and 0.01 M ammonium acetate (pH 5.0) and detected at UV wavelength of 280 nm.
Monometoxytrityl derivatives of uridine as inhibitors of a human recombinant UDP-glucuronosyltransferase : UGT1*6
1997, Life Sciences
A series of inhibitors of the human liver recombinant UDP-glucuronosyltransferase 1^∗6 derived from uridine were synthetized as probes of the binding site of the cosubstrate, UDP-glucuronic acid. If triphenylmethanol or uridine alone failed to inhibit the glucuronidation of 4-methylumbelliferone, the trityl derivatives of uridine were found to be very effective inhibitors of the enzyme (Ki 4.4 to 73 μM). The type of inhibition (competitive or mixed) varied with the substitutions on the uracile or on the triphenylmethyl moiety by halogen atoms or methyl groups. Structural features for the binding of the cofactor are postulated.
The influence of age and some inducers on UDP-glucuronyltransferase activity
1997, Experimental Gerontology
UDP-glucuronyltransferase (UDP-GT) activity was examined in male Wistar rats aged 0.5, 1, 2, 4, 8, 12, 20, and 28 months. The rats were treated with phenobarbital (75 mg/kg, 72 and 48 h before death), β-naphthoflavone or dexamethasone (40 mg/kg and 20 mg/kg, respectively, for three days before death). Prior to decapitation the rats were fasted for 12 h. Hepatic microsomes were prepared according to the method of Dallner. UDP-GT activity was determined by the method of Burchell and Weatherill. p-Nitrophenol was used as an aglucone. UDP-GT activity decreased rapidly in the control rats aged from two weeks to four months. In the older control rats the activity tended to increase. Two-week-old rats treated with phenobarbital showed a slightly increased UDP-GT activity. In the older animals (up to one year) UDP-GT activity increased to 150% of the control value and stayed at this level in the remaining age groups. β-Naphthoflavone was a more potent inducer of UDP-GT than phenobarbital. The activity of β-naphthoflavone-induced UDP-GT was low in the youngest rats. It was about 180% in two-month-old rats and reached 260% of the control value in eight-month-old rats. Although the activity decreased in the older rats, it still exceeded 200%. Dexamethasone did not affect UDP-GT activity. Only in two-week-old and two-month-old rats did we observe a slight increase in the activity of UDP-GT.

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^☆: This work was supported, in part, by a grant from the “Ministère de la Recherche et de la Technologie” (n∘ 88 T 0813).

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Phenobarbital inducible UDP-glucuronosyltransferase is responsible for glucuronidation of 3′-azido-3′-deoxythymidine: Characterization of the enzyme in human and rat liver microsomes☆

Abstract

Biochem Pharmacol

Comp. Biochem. Physiol

Anal. Biochem

J. Biochem. Biophys. Methods

J. Biol. Chem

Biochem. Pharmacol

Biochem. Pharmacol

Biochem. Pharmacol

Chemomet. Intel. Lab. Sys

Eur. J. Med. Chem

Comp. Biochem. Physiol

J. Pharmac. Clin