Mono- and diglucuronide formation from benzo[a]pyrene and chrysene diphenols by AHH-1 cell-expressed UDP-glucuronosyltransferase UGT1A7

doi:10.1016/S0006-2952(98)00337-2

Biochemical Pharmacology

Volume 57, Issue 6, 15 March 1999, Pages 653-656

https://doi.org/10.1016/S0006-2952(98)00337-2 Get rights and content

Abstract

Polycyclic aromatic hydrocarbon (PAH)-type compounds induce at least two rat UDP-glucuronosyltransferase isoforms, UGT1A6 and UGT1A7. Among the glucuronidation reactions of PAH metabolites studied, mono- and diglucuronide formation of benzo[a]pyrene and chrysene-3,6-diphenol showed the highest induction factors in rat liver microsomes. Availability of AHH-1 cells stably expressing UGT1A7 allowed us to study whether this PAH-inducible isoform could catalyze benzo[a]pyrene and chrysene-3,6-diphenol glucuronidation. It was found that UGT1A7 indeed catalyzed mono- and diglucuronide formation of both benzo[a]pyrene and chrysene 3,6-diphenols. V79 cell-expressed rat UGT1A6 also catalyzed these reactions, except for chrysene diphenol diglucronide formation (Bock et al., Mol Pharmacol 42: 613–618, 1992). Enzyme kinetic studies of the glucuronidation of 6-hydroxychrysene (used as a stable PAH phenol) indicated that UGT1A7 conjugated this compound with a lower apparent K_m value (0.1 μM) than UGT1A6 (10 μM). The results suggest that the two PAH-inducible UGTs may cooperate in conjugating PAH metabolites, but that UGT1A7 is more efficient.

Section snippets

Chemicals

6-Hydroxychrysene and chrysene-3,6-diphenol were synthesized as described [12] and identified by high-field proton NMR spectroscopy and mass spectrometry. Their purity was found to be >98%, based on HPLC analysis. BP-3,6-quinone was obtained from the Carcinogen Reference Standard Repository, National Institutes of Health (Bethesda, MD, USA).

Cells

UGT1A7-transfected AHH-1 cells were grown in culture flasks containing 50 mL RPMI 1640 medium including 1% (w/v) penicillin/streptomycin, 10% (v/v) horse

Results and discussion

MG and DG formation of both BP- and chrysene-3,6-diphenol could be clearly demonstrated in incubation mixtures containing lysates of UGT1A7-expressing cells (Table 1). Formation of these glucuronides has also been observed in UGT1A6-expressing V79 cells, with the notable exception that chrysene-3,6-diphenol DG formation was not detectable [12]. Kinetic characteristics were determined with 6-hydroxychrysene as a stable PAH phenol and compared with those found with a widely used model substrate,

Acknowledgements

The authors wish to thank the Deutsche Forschungsgemeinschaft (DFG) for financial support.

References (22)

A.D. Grove et al.
Identification of a rat oltipraz-inducible UDP-glucuronosyltransferase (UGT1A7) with activity towards benzo[a]pyrene-7,8-dihydrodiol
J Biol Chem
(1997)
M.D. Burke et al.
Metabolism of benzo[a]pyrene with isolated hepatocytes and the formation and degradation of DNA-binding derivates
J Biol Chem
(1977)
K.W. Bock et al.
Release of mutagenic metabolites of benzo[a]pyrene from the perfused rat liver after inhibition of glucuronidation and sulfation by salicylamide
Chem-Biol Interact
(1981)
C. Legraverend et al.
Bone marrow toxicity induced by oral benzo[a]pyreneProtection resides at the level of the intestine and liver
Toxicol Appl Pharmacol
(1983)
S.-I. Ikushiro et al.
Identification and analysis of drug-responsive expression of UDP-glucuronosyltransferase family 1 (UGT1) isozyme in rat hepatic microsomes using anti-peptide antibodies
Arch Biochem Biophys
(1995)
K.W. Bock et al.
Paracetamol glucuronidation by recombinant rat and human phenol UDP-glucuronosyltransferases
Biochem Pharmacol
(1993)
O.H. Lowry et al.
Protein measurement with the Folin phenol reagent
J Biol Chem
(1951)
E.C. Miller et al.
Searches for ultimate chemical carcinogens and their reactions with cellular macromolecules
Cancer
(1982)
A.H. Conney
Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic aromatic hydrocarbonsG.H.A. Clowes memorial lecture
Cancer Res
(1982)
D.R. Bevan et al.
Quinol diglucuronides are predominant conjugated metabolites found in bile of rats following intratracheal instillation of benzo[a]pyrene
Carcinogenesis
(1992)

K.W. Bock et al.

Absorption and metabolism of naphthalene and benzo[a]pyrene in the rat jejunum in situ

Med Biol

(1979)

Cited by (17)

A comprehensive review of UDP-glucuronosyltransferase and esterases for drug development
2015, Drug Metabolism and Pharmacokinetics
Citation Excerpt :
UGT1A7: UGT1A7, UGT1A8, UGT1A9, and UGT1A10 are characterized by the high homology of their first exons. UGT1A7 exhibits glucuronidation activity toward various carcinogens, such as hydroxylated benzo[a]pyrenes and N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine [137,138]. This enzyme also catalyzes the glucuronidation of SN-38 and mycophenolic acid [139,140].
UDP-glucuronosyltransferase (UGT) and esterases are recognized as the most important non-P450 enzymes because of their high contribution to drug metabolism. UGTs catalyze the transfer of glucuronic acid to hydroxyl, carboxyl, or amine groups of compounds, whereas esterases hydrolyze compounds that contain ester, amide, and thioester bonds. These enzymes, in most cases, convert hydrophobic compounds to water-soluble metabolites to facilitate the elimination of compounds from the body. Information about these enzymes is steadily increasing, although our knowledge is still behind our understanding of P450. This review gives an overview of recent findings in UGT and esterases studies focusing on tissue distribution, gene regulation, substrate and inhibitor specificity, and species differences. In particular, the absolute protein content of UGT isoforms and esterases in human tissues could be available. In the field of esterases, it is becoming clear that enzymes other than carboxylesterase are involved in drug hydrolysis. In addition, there is an interesting interplay between UGTs and esterases in the formation and hydrolytic deglucuronidation of acyl-glucuronide, which is considered to be a reactive metabolite. With the growing awareness of the importance of non-P450 enzymes in drug development, issues that should be resolved are discussed.
Topological aspects of oligomeric UDP-glucuronosyltransferases in endoplasmic reticulum membranes: Advances and open questions
2009, Biochemical Pharmacology
UDP-glucuronosyltransferases (UGTs) represent major Phase II enzymes involved in detoxification of endo- and xenobiotics, including many drugs. The intraluminal orientation of the active site of UGTs in endoplasmic reticulum membranes necessitates a number of transporters in these membranes, for example, for UDP-glucuronic acid and glucuronides, the latter being insufficiently characterized. In addition, accumulating evidence suggests that UGTs are functional as homo- and heterodimers in monoglucuronide formation. They may form tetramers in diglucuronide formation. UGT oligomers probably serve to stabilize UGT monomers and fine-tune UGT activity. Glucuronide disposition may also be influenced by endoplasmic reticulum-localized β-glucuronidase, possibly involved in hydrolysis of hormone and drug glucuronides in target cells. The present commentary reviews recent advances and addresses open questions. Resolution of these questions may help to understand many problems of glucuronide synthesis and disposition in vivo, for example, under-prediction of the in vivo clearance of drugs mostly eliminated by glucuronidation by in vitro enzyme kinetic parameters of UGTs.
Coordinate regulation of Phase I and II xenobiotic metabolisms by the Ah receptor and Nrf2
2007, Biochemical Pharmacology
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor with important roles in metabolic adaptation, normal physiology and dioxin toxicology. Metabolic adaptation is based on coordinate regulation of a set of xenobiotic-metabolizing enzymes (XMEs), termed AhR battery. Coordination is achieved by AhR/Arnt-binding to XREs (xenobiotic response elements), identified in the 5′ upstream region of AhR target genes. The AhR battery encodes Phase I and II enzymes. Interestingly, these Phase II genes are linked to the Nrf2 gene battery that encodes enzymes that are essential in protection against oxidative/electrophile stress. Nrf2 binds to AREs (antioxidant response elements) in the regulatory region of a large and distinct set of target genes. Functionally characterized response elements such as XREs and AREs in the regulatory region of target genes may provide a genetic basis to understand AhR- and Nrf2-induced genes. Linkage between AhR and Nrf2 batteries is probably achieved by multiple mechanisms, including Nrf2 as a target gene of the AhR, indirect activation of Nrf2 via CYP1A1-generated reactive oxygen species, and direct cross-interaction of AhR/XRE and Nrf2/ARE signaling. Linkage appears to be species- and cell-dependent. However, mechanisms linking XRE- and ARE-controlled Phase II genes need further investigation. Tightened coupling between Phases I and II by AhR- and Nrf2-induced XMEs may greatly attenuate health risks posed by CYP1A1-generated toxic intermediates and reactive oxygen species. Better recognition of coordinate Phase I and II metabolisms may improve risk assessment of reactive toxic intermediates in the extrapolation to low level endo- and xenobiotic exposure.
Induction of gene expression of xenobiotic metabolism enzymes and ABC-transport proteins by PAH and a reconstituted PAH mixture in human Caco-2 cells
2004, Biochimica et Biophysica Acta - Gene Structure and Expression
It was shown recently that in epithelial Caco-2 cells the food contaminant benzo[a]pyrene (B[a]P) is metabolized and B[a]P-sulfate metabolites were transported out of the cells. The aim of this study was to investigate whether B[a]P and other polycyclic aromatic hydrocarbons (PAH) such as chrysene, phenanthrene, benzo[k]fluoranthene (B[k]F), dibenzo[a,l]pyrene (DB[a,l]P), and pyrene alone or in a mixture in a ratio as they occur in tobacco smoke have effects on gene expression of intestinal cytochrome P450 enzymes (CYP), Phase II enzymes and ATP-binding cassette (ABC)-transport proteins in the human Caco-2 cells. B[a]P induced its own metabolism. Treatment of the Caco-2 cells with B[a]P, chrysene, B[k]F, or DB[a,l]P induced mRNA expression of CYP1A1 and CYP1B1 specifically as measured by RT-PCR. In contrast, the mRNA expression of the microsomal epoxide hydrolase (mEH) was not affected by PAH. The gene expression of the Phase II enzymes UDP-glucuronosyltransferase 1A6 (UGT1A6) and UGT1A7 was also induced by these PAH but treatment with them had no effect on gene expression of sulfotransferases (SULT) at all. Of the ABC-transport proteins, MDR1 mRNA expression was induced by treatment with carcinogenic PAH, whereas MRP2 mRNA expression was not changed. The mixture of PAH also induced CYP1A1, CYP1B1, UGT1A6, and UGT1A7 mRNA expression. We conclude that B[a]P, chrysene, B[k]F, and DB[a,l]P have specific effects on intestinal CYP1A1, CYP1B1, UGT1A6, and UDP1A7 mRNA expression but no effects on the expression of SULT.
An alternative promoter contributes to tissue- and inducer-specific expression of the rat UDP-glucuronosyltransferase 1A6 gene
2001, Toxicology and Applied Pharmacology
UDP-glucuronosyltransferase 1A6 (UGT1A6), a key enzyme catalyzing the glucuronidation of small planar phenols and amines, is expressed in a tissue- and inducer-dependent manner. Expression is high in kidney, gastrointestinal tract, and induced liver, with low expression in spleen, lung, and ovary. Exposure to certain chemicals, such as 3-methylcholanthrene, benzo[a]pyrene, β-naphthoflavone, and oltipraz elevates UGT1A6 mRNA in liver and to a lesser extent gastrointestinal tract and kidney, but not in other tissues. The mechanisms underlying this complex pattern of expression have been elusive. We have identified a new type of UGT1A6 mRNA (class 2) that differs in its 5′ untranslated sequence. The class 2 transcript is the more abundant type expressed in liver, gastrointestinal tract, and kidney. Transcription of the class 2 mRNA is initiated 107 bases 5′ of the UGT1A6 coding exon. The promoter region flanking the transcription start site contains an HNF1-like binding site identical to that in the human UGT1A6 gene. Both class 1 and class 2 mRNAs were elevated in liver by 3-methylcholanthrene, benzo[a]pyrene, β-naphthoflavone, and oltipraz, with preferential elevation of class 1 occurring after 3-methylcholanthrene and benzo[a]pyrene treatment. These data suggest that transcription from a second promoter contributes to tissue- and inducer-specific expression of rat UGT1A6.
Differential protection by rat UDP-glucuronosyltransferase 1A7 against benzo[a]pyrene-3,6-quinone-versus benzo[a]pyrene-induced cytotoxic effects in human lymphoblastoid cells
2000, Toxicology and Applied Pharmacology
UDP-glucuronosyltransferase 1A7 (UGT1A7) is a polyaromatic hydrocarbon (PAH)–inducible UGT with activity toward various benzo[a]pyrene (B[a]P) metabolites. To investigate the influence of rat UGT1A7 on B[a]P-induced cytotoxicity, human lymphoblastoid L3 cells were transfected with pMF6 (control expression vector), p167Dtk2 (microsomal epoxide hydrolase expression vector), or p167Dtk2-1A7 (epoxide hydrolase/UGT1A7 coexpression vector), and the cell populations were compared for sensitivity to B[a]P-induced effects. B[a]P inhibited cell proliferation and decreased relative cell survival of p167Dtk2 and p167Dtk2-1A7 cells to a similar extent. Metabolism studies using [³H]B[a]P revealed increased formation of glucuronide conjugates of B[a]P-4,5-diol, 3-OH-, or 9-OH-B[a]P and an unidentified metabolite by p167Dtk2-1A7 cells, but the presence of unconjugated metabolites suggested that glucuronidation capacity may be limited. No differences between p167Dtk2 and p167Dtk2-1A7 L3 cells were observed in the growth inhibitory effects of 3-OH-B[a]P or B[a]P-7,8-diol, but p167Dtk2-1A7-expressing cells were found to be less sensitive to B[a]P-3,6-quinone-induced effects on cell proliferation and relative cell survival. The effect was also observed in AHH-1 lymphoblastoid cells expressing UGT1A7 without epoxide hydrolase. The UGT1A7-expressing AHH-1 cells were also less sensitive to growth inhibition by B[a]P-1,6-quinone and B[a]P-6,12-quinone. Flow cytometric analysis of vehicle and B[a]P-3,6-quinone-exposed cell populations showed an association between UGT1A7 expression and resistance to B[a]P-3,6-quinone-induced apoptosis and loss of cell viability. These data suggest that UGT1A7 may be preferentially active toward B[a]P-quinones and that UGT1A7 may represent the PAH-inducible UGT activity previously implicated in protection against toxic redox cycling by B[a]P-3,6-quinone.

View all citing articles on Scopus

View full text

Molecular and Cellular PharmacologyMono- and diglucuronide formation from benzo[a]pyrene and chrysene diphenols by AHH-1 cell-expressed UDP-glucuronosyltransferase UGT1A7

Abstract

Section snippets

Chemicals

Cells

Results and discussion

Acknowledgements

J Biol Chem

J Biol Chem

Chem-Biol Interact

Toxicol Appl Pharmacol

Arch Biochem Biophys

Biochem Pharmacol

J Biol Chem

Searches for ultimate chemical carcinogens and their reactions with cellular macromolecules

Cancer

Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic aromatic hydrocarbonsG.H.A. Clowes memorial lecture

Cancer Res

Quinol diglucuronides are predominant conjugated metabolites found in bile of rats following intratracheal instillation of benzo[a]pyrene

Carcinogenesis

Absorption and metabolism of naphthalene and benzo[a]pyrene in the rat jejunum in situ

Med Biol

Molecular and Cellular Pharmacology
Mono- and diglucuronide formation from benzo[a]pyrene and chrysene diphenols by AHH-1 cell-expressed UDP-glucuronosyltransferase UGT1A7