The contribution of UDP-glucuronosyltransferase 1A9 on CYP1A2-mediated genotoxicity by aromatic and heterocyclic amines

Mei-Fei Yueh, Nghia Nguyen, Maryam Famourzadeh, Christian P. Strassburg1,, Yoshimitsu Oda2,, F.Peter Guengerich3, and Robert H. Tukey4,

Departments of Chemistry, Biochemistry and Pharmacology, University of California, San Diego, La Jolla, CA 92093-0636, USA,
1 Department of Gastroenterology and Hepatology, Hannover Medical School, 30625 Hannover, Germany,
2 Osaka Prefectural Institute of Public Health, 3-69 Nakamichi 1-chome, Higashinari-ku, Osaka 537, Japan and
3 Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt School of Medicine, Vanderbilt University, Nashville, TN 37232-0146, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
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The importance of environmental and dietary arylamines, and heterocyclic amines in the etiology of human cancer is of growing interest. These pre-carcinogens are known to undergo bioactivation by cytochrome P450 (CYP)-directed oxidation, which then become substrates for the UDP-glucuronosyltransferases (UGTs). Thus, glucuronidation may contribute to the elimination of CYP-mediated reactive intermediate metabolites, preventing a toxic event. In this study, human UGTs were analyzed for their ability to modulate the mutagenic actions of N-hydroxy-arylamines formed by CYP1A2. Studies with recombinant human UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7 and UGT2B15 expressed in heterologous cell culture confirmed that UGT1A9 glucuronidated the mutagenic arylamines N-hydroxy-2-acetylaminofluorene (N-hydroxy-2AAF) and 2-hydroxyamino-1-methyl-6-phenylimidazo(4,5-b)pyridine (N-hydroxy-PhIP). To examine the mutagenic potential of these agents, a genotoxicity assay was employed using Salmonella typhimurium NM2009, a bacterial strain expressing the umuC SOS response gene fused to a ß-galactosidase reporter lacZ gene. DNA modification results in the induction of the umuC gene and subsequent enhancement of ß-galactosidase activity. Both N-hydroxy-2AAF and N-hydroxy-PhIP stimulated a dose-dependent increase in bacterial ß-galactosidase activity. In addition, the procarcinogens 2AAF and PhIP were efficiently bioactivated to bacterial mutagens when incubated with Escherichia coli membranes expressing CYP1A2 and NADPH reductase. CYP1A2 generated 2AAF- and PhIP-mediated DNA damage, but only the action of N-hydroxy-2AAF was blocked by expressed UGT1A9. These results indicate that UGT1A9 can control the outcome of a genotoxic response. The results also indicate that while a potential toxicant such as N-hydroxy-PhIP can serve as substrate for glucuronidation, its biological actions can exceed the capacity of the detoxification pathway to prevent the mutagenic episode.

Abbreviations: 2AAF, 2-acetylaminofluorene; CYP, cytochrome P450, S. typhyimurium, Salmonella typhimurium; N-hydroxy-2AAF, N-hydroxy-2-acetylaminofluorene; N-hydroxy-PhIP, 2-hydroxy-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine; ß-NAPD, ß-nicotinamide adenine dinucleotide phosphate; ONGP, O-nitrophenyl-ß-D-galactophranoside; PhIP, 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine; Sf9, Spodoptera frugiperda insect cells; UDPGlcA, uridine 5'-diphosphoglucuronic acid; UGT, UDP-glucuronosyl-transferase.


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 Abstract
 Introduction
 Materials and methods
 Results
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The neoplastic transformation of epithelial cells by chemical carcinogens has been suggested in cancers linked to the esophagus, stomach, bladder, liver, colon, lung and pleura. While there is convincing evidence that the initiation of cancer following exposure to environmental toxicants can be attributed to patterns of metabolic activation of pre-carcinogens by microsomal oxidation, detoxification pathways may also play a significant role in the metabolic defense against environmental mutagens aimed at facilitating their removal from the cells prior to initiation of the mutagenic event. The Phase II families of enzymes involved in conjugation reactions participate in cellular trapping and elimination of potentially harmful metabolic by-products and xenobiotics by forcing the conjugates into the aqueous compartments enabling elimination through kidney or biliary excretion. The UDP-glucuronosyltransferases (UGTs), which as a multigene family share a wide range of substrate specificities (1), have been proven to participate in the conjugation of potentially harmful oxidative products. Recent evidence has indicated that early down-regulation of the UGTs in premalignant and malignant tumor tissues but not benign tumors may be a prerequisite of cellular carcinogenesis (2), indicating that the deregulation of the metabolic and cellular defense mechanisms compromises the normal homeostatic functions of the cell.

At present, 15 human UGTs have been cloned and most have been characterized in terms of functional properties through expression in heterologous cellular systems (1). Along with the cloning and characterization of the human P450s (4), biological tools are now available to design expression systems to study the contribution of individual UGTs in the metabolism of potentially harmful oxidative products catalyzed by the CYPs. One class of compounds that contains excellent candidates for the formation of oxidative derived mutagens as well as potential substrates for glucuronidation is the aromatic and heterocyclic amines (arylamines).

Arylamines represent a class of potent bacterial mutagens and are believed to be animal and human carcinogens (57). N-Hydroxylation of aromatic amines is also correlated with DNA adduct formation in animal studies (8,9), and has been shown to initiate chemical mutagenesis in the classical reverse mutation assay described by Ames et al. Oxidative metabolism bioactivates arylamines to potential mutagens through N-hydroxylation (1013), followed by esterification by acetyltransferases and sulfotransferases, a process which facilitates DNA binding and mutagenesis. We and others have demonstrated that these compounds gain their biological activity through metabolic conversion by oxidative bioactivation which is initiated primarily by microsomal CYP1A2 (13,1420).

Our laboratory has identified and cloned the human UGT1A cDNAs as well as UGT2B4, UGT2B7, UGT2B10 and UGT2B15 (21), by taking advantage of their unique expression patterns in tissues of the hepatocellular–gastrointestinal tract (3,2224). Data generated from cDNA-directed heterologous expression of the human cDNAs have demonstrated that several UGTs, primarily UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9 and UGT1A10, are capable of catalyzing the glucuronidation of aromatic and heterocyclic amines (1). Glucuronides of the food-borne heterocyclic amines, which can be generated using human hepatic microsomal protein (25) or recombinant UGT protein (26), have also been isolated in human urine. We have recently demonstrated that N-hydroxy-PhIP serves as a substrate primarily for expressed UGT1A9 (21,26). The potential thus exists that carcinogenic arylamines formed through CYP1A2 dependent N-hydroxylation might become predisposed as substrates for glucuronidation, a process that would decrease their carcinogenic potential.

In this study, the N-acylarylamine 2-AAF and the heterocyclic amine PhIP were used as model compounds to examine UGT metabolism following activation through N-hydroxylation. 2-AAF is among the most intensively studied of all genotoxins (11,27,28), while PhIP and other heterocyclic amines are formed in protein-rich foods as a result of pyrolysis during cooking (2931). To examine the impact of CYP1A2-directed bioactivation and the role of glucuronidation, experiments were designed to detect the alteration of mutagenicity by incorporation of expressed CYP1A2 and the UGTs in a single catalytic reaction. In order to avoid the confounding effects of other xenobiotic-metabolizing enzymes, S9 fractions were prepared from recombinant protein expression for both CYP1A2 and UGT proteins. A mutagenicity assay was employed that uses Salmonella typhimurium strain NM2009, which carries a chimera umuC'lacZ (32,33). In this assay the umuC gene, controlled by the lexA and recA genes of the bacterial SOS response gene, is inducible by DNA-damaging alkylating agents. The SOS response is stimulated following proteolytic cleavage of LexA protein by activated RecA protease, a result that leads to induction of ß-galactosidase production. In this model, S.typhimuruim NM2009 has been used successfully to detect carcinogens in environmental samples (34,35) as well as study the ability of human CYP proteins to bioactivate a diverse array of procarcinogens (3640) including arylamines (4144). The ability to produce active CYP1A2 as well as the different UGTs allowed us to study the modulating role of UGT in genotoxicity of N-hydroxyarylamines.


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Reagents
2-AAF, N-hydroxy-2AAF and PhIP were obtained from the National Cancer Institute Chemical Carcinogen Repository, Midwest Research Institute (Kansas City, MO). N-Hydroxy-PhIP was from Toronto Research Chemicals (Toronto, Canada). ONGP, UDPGLcA, D-glucose-6-phosphate, ß-NAPD, D-glucose-6-phosphate dehydrogenase and substrates used for analysis of UGT activity were purchased from Sigma (St Louis, MO). Horseradish peroxidase conjugated secondary antibody and the `Supersignal' chemiluminescent detection kit (for immunoblotting) were from Pierce (Rockford, IL). Bio-Rad Protein Assay for protein concentration analysis was purchased from Bio-Rad (Hercules, CA). Thin layer chromatography plates were from Whatman (Clifton, NJ). Tris–glycine gels for SDS–PAGE were from Novex (San Diego, CA). pcDNA expression vector was purchased from Invitrogen (Carlsbad, CA).

Production of recombinant baculovirus and heterologous protein expression
Full length UGT cDNAs encoding UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A9, UGT1A10, UGT2B4, UGT2B7 and UGT2B15, cloned from our laboratory using different human tissue RNA (21,23,45), were subcloned into the baculovirus expression plasmid pBlueBac4.5 (Invitrogen), and the selection of high titer recombinant baculovirus produced in Sf9 cells as previously described (46). Since our initial attempt to clone UGT1A8 resulted in the production of an inactive recombinant UGT (23), this UGT was recloned from human colon RNA, subcloned into the baculovirus pFASTBAC expression vector (Life Technologies, Gaithersburg, MD) and expressed using the Bac to Bac expression system using instructions outlined by the vendor. pFASTBAC UGT1A8 is catalytically active toward small phenolic substrates such as 4-methylumbelliferone.

Each of the full length UGT1A and UGT2B cDNAs were also transferred to the expression vector pcDNA3.1-neo (Invitrogen) for selection and expression in either human HK293 cells or mouse ovary V79 cells. The recombinant pcDNA3.1-UGTs were transfected into these cells using the PerFect Lipid Tranfection kit (Invitrogen) followed by treatment with 800 µg/ml neomycin for several weeks. Once clones were established, they were individually selected and the cells plated at low density in the presence of neomycin to allow for one more round of clonal selection. Each of the HK293 and V79 clones were tested for UGT activity using total cell extracts.

Immunoblot analysis
Expressed UGT proteins were identified by immunoblot analysis. The cell pellet was suspended in 5 vol 50 mM Tris–HCl buffer, pH 7.2, containing 10 mM MgCl2 and 1 mM phenylmethysulfonyl fluoride, and the cells were sonicated for 5x5s. The cell lysate was pelleted at 10 000 g for 10 min at 4°C. The cells were re-suspended in the same buffer, sonicated for 5x5s, and pelleted at 10 000 g for 10 min at 4°C. The two supernatants were pooled and centrifuged at 100 000 g for 60 min at 4°C. The microsomal pellet was suspended in 50 mM Na2HPO4 buffer, pH 7.4, containing 0.1 mM EDTA and 10% (v/v) glycerol. Protein concentrations were determined using a Bio-Rad Protein Assay with bovin serum albumin as a standard. Microsomal proteins were separated by electrophoresis in a 10% (v/v) polyacrylamide pre-cast Tris–glycine gel and the resolved proteins were transferred to nitrocellulose. Detection of UGT proteins was accomplished with a primary anti-human UGT1A antiserum raised in rabbits against a 15mer UGT1A peptide fragment [residues 441 and 455 on exon 5 of the constant C-terminal portion of the UGT1A proteins (23)]. The secondary antibody was horseradish peroxidase-conjugated goat anti-rabbit IgG. Visualization was achieved by chemiluminesence using the `Supersignal' kit from Pierce.

Identification of human UGT activities
UGT activities were determined by TLC assay according to the method of Bansal and Gessner (47). Cells expressing the recombinant UGTs were collected and homogenized in 5 vol lysis buffer. Each UGT assay was conducted in a total volume of 100 µl containing 50 mM Tris–HCl (pH 7.6), 10 mM MgCl2, 500 µM UDPGlcA, 0.04 µCi [14C]UDPGlcA (0.14 nmol), 8.5 mM saccharolactone, 100 µM substrate (dissolved in methanol) and 100 µg protein from the different cell extracts [final methanol concentration did not exceed 5% (v/v)]. Each reaction was incubated at 37°C for 30 min and then stopped by the addition of 100 µl ethanol. The protein was removed by centrifugation at 10 000 r.p.m. for 10 min in an Eppendorf centrifuge, and 100 µl supernatant was spotted on 250 µm Whatman glass-back silica gel TLC plates. TLC plates were developed with a mixture of 35:35:10:20 (v/v) n-butanol/acetone/acetic acid/water. Each TLC plate was dried and visualized with a Storm 820 PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Resident glucuronides were then removed and quantitated by liquid scintillation counting. Km determinations with N-hydroxy-2AAF (for UGT1A9) were conducted with a range of substrate concentrations: 1, 2, 4, 10, 20, 40, 100, 200 and 400 µM and the values plotted using Lineweaver–Burk analysis. Catalytic activities were expressed as absolute values, which does not adjust the activities for the levels of expressed UGT, or as relative values, which takes into consideration UGT protein abundance.

UmuC genotoxicity assay
Human CYP1A2 was co-expressed with NADPH-P450 reductase in Escherichia coli using a bicistronic system (pCW'1A2:NPR) in which a single mRNA is produced and is translated into two proteins (48). The bacterial tester strain, S.typhimurium NM2009 (TA1535/pSK1002/pNM12) carried a fused umuC'lacZ gene. DNA damage by genotoxins induces expression of the umuC gene, a gene controlled by SOS response (49), and subsequently results in expression of ß-galactosidase, encoded by the reporter lacZ gene. Additionally, the bacterial O-acetyltransferase gene (in pNM plasmid) was transformed into the original S.typhimurium TA1535/pSK1002 strain, yielding a system that is highly sensitive to the mutagenic actions of heterocyclic/aromatic amines by generation of N-acetoxy metabolites, which subsequently break down into nitrenium ions (43,44). In this model, the genotoxicity of the procarcinogen was dependent upon human CYP1A2 which had been expressed E.coli, as measured using umuC gene expression. The genotoxicity assay was carried out according to the procedure described by Shimada et al. (33). Briefly, an overnight culture of bacteria tester strain (NM2009) was diluted 40-fold into TGA medium (1% (w/v) bacto-tryptone, 0.2% (w/v) glucose, 0.5% (w/v) NaCl, 25 µg/ml ampicillin, 10 µg/ml chloramphenicol) until the bacteria density reached an OD600 of 0.25–0.3. To achieve the maximum efficiency of metabolic activation by CYP1A2, membrane-containing CYP1A2 was pre-incubated with test compound at 37°C for 20 min, followed by addition of 1.7 ml tester bacteria. The pre-incubation mixture (300 µl) included 100 µM potassium phosphate buffer (pH 7.4), E.coli membrane fraction (40–100 µg) containing CYP1A2/NADPH reductase, NADPH generating system (5 mM glucose-6-phosphate, 1 mM ß-NADP and 1 U glucose-6-phosphate dehydrogenase) and procarcinogen dissolved in 10 µl DMSO. After 2 h vigorously shaking at 37°C, the ß-galactosidase assay for mutagenic activity was examined. The bacterial density were measured at OD600 and was used for both normalization of expression levels and indication of cellular cytotoxicity. The genotoxicity data was taken only at OD600 > 60% of control group to eliminate cytotoxic effects. A fraction (0.1 ml) of the culture was diluted with 0.9 ml Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4 pH 7.0, 10 mM KCl, 1 mM MgSO4, 50 mM 2-mercaptoethanol) and the bacterial cells were made permeable to the chromogenic substrate for ß-galactosidase by adding 0.1% SDS and one drop of chloroform, then mixing vigorously. The enzyme reaction was initiated by the addition of 0.1 ml 2-nitrophenyl-ß-D-galactopyranoside solution (4.0 mg/ml in 0.1 M sodium phosphate buffer, pH 7.0) and incubated at 37°C for 15 min. The reaction was stopped by adding 0.5 ml Na2CO3 and the absorbance at OD420 and OD550 was measured (32).

The ability of UGT metabolism to interrupt the mutagenicity of arylamine metabolites was examined by directly adding activated mutagen (N-hydroxy 2AAF or N-hydroxy PhIP) to a 300 µl reaction mixture containing 50 mM MgCl2, 10 mM Tris–HCl pH 7.2, 500 µM UDPGLcA, 8.5 mM saccharolactone and 100 µg expressed UGT from cell lysates. Following a 60 min pre-incubation, the reaction mixture was centrifuged to eliminate cellular mass and the supernatant was added to 1.7 ml bacterial tester strain, then shaken at 37°C for 2 h. umuC gene expression was estimated from ß-galactosidase activity. All umuC assays were performed at least twice with duplicate tubes at each concentration of substrate.


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Catalytic activities of expressed guts
Recombinant proteins from heterologous expression systems, including Sf9 expressed recombinant baculovirus or stable expression in HK293 or V79 cells were generated for human UGT1A1, -1A3, -1A4, -1A6, -1A7, -1A8, -1A9, -1A10, -2B4, -2B7 and -2B15. Each of the different extracts were prepared to a concentration of ~5 mg/ml protein and the expressed enzymes were evaluated with the intention of obtaining the most efficient expression system for each UGT. While each cellular model displayed catalytic activity, we found greater catalytic activity when UGT1A4, UGT1A6, UGT1A9 and UGT2B4 were expressed in HK293 cells and UGT1A1, UGT1A3, UGT1A7, UGT1A8 and UGT1A10 were expressed in V79 cells (Table IGo). Baculovirus infected insect Sf9 cells expressed UGT2B7 and UGT2B15 more efficiently than their expression in HK293 and V79 cells. Expressed UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4 and UGT2B7 were all active with substrates that have previously been determined to elicit maximal glucuronidation activity (1). Examples of activities with control substrates are shown in Table IGo. While significant protein can be produced within Sf9 cells infected with a UGT2B15 construct, the extracts express little UGT activity, even with 4-methylumbilliferone, which had been shown previously to be a substrate for expressed UGT2B15 in HK 293 cells (50). Thus, UGT2B15 was not used in additional experiments. The cellular preparations that were employed in these studies expressed the different UGTs, as demonstrated by immunoblot analysis (Figure 1Go). Also shown is an approximation of the relative expression levels, which were determined based upon the fraction equivalent of the most abundant UGT (UGT1A8). Taking these values into consideration, a second set of catalytic activities were generated as shown in Table IGo. While these relative expression values are important, it should be noted that we did not attempt to dilute the cellular preparations for catalytic analysis based upon the outcome of protein abundance as judged by western blot analysis. Since these are clonal cell lines, we felt it more important that the activities be represented based upon total cellular protein, since this accurately reflects the abundance levels of each of our characterized cell lines.


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Table I. UDP-glucuronosyltransferase activities of the expressed UGTs using a series of control substrates and N-hydroxy-2AAF (N-OH-2AAF) and N-hydroxy-PhiP (N-OH-PhIP)a
 


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Fig. 1. Recombinant UGT1A proteins recognized by immunoblot analysis. Microsomal proteins from cell lines expressing the human UGT proteins were separated by electrophoresis using 10% SDS–PAGE and the proteins were transferred to nitrocellulose. Detection of the expressed UGTs was accomplished with an anti-peptide UGT1A antibody previously developed in this laboratory 24. (Top) Enhanced chemiluminescence imaging. (Bottom) Relative expression levels based upon the expression of UGT1A8. Not shown are the abundance levels of UGT2B4, UGT2B7 or UGT2B15.

 
Ability of expressed UGTs to glucuronidate N-hydroxy-2AAF and N-hydroxy-PhIP
Examination of N-hydroxy-2AAF demonstrated that the primary UGTs involved in the glucuronidation of this substrate were UGT1A7 or UGT1A9 (Table IGo). While UGT1A3, UGT1A6, UGT1A8, UGT1A10 and UGT2B7 were also active toward N-hydroxy-2AAF, their activities were considerably lower than that for UGT1A7 and UGT1A9. Using extracts for expressed UGT1A7 and UGT1A9, Figure 2Go shows the kinetics of N-hydroxy-2AAF glucuronide formation, using a range of substrates from 1 to 400 µM. The UGT1A9 reaction was characterized by a Km of 15 µM and a Vmax of 370 pmol/min/mg protein. In comparison, UGT1A7 generated N-hydroxy-2AAF glucuronide with ~30% the rate, 138.6 pmol/min/mg protein.



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Fig. 2. Kinetic analysis of N-hydroxy 2-AAF glucuronidation catalyzed by UGT1A9. Assays were performed with the substrate N-hydroxy 2-AAF (1, 2, 4, 10, 20, 40, 100, 200 and 400 µM) as described in Materials and methods.

 
In previous experiments, our laboratory determined that N-hydroxy-PhIP was glucuronidated by baculovirus-expressed UGT1A9 (21), with activity also expressed by UGT1A7 and UGT1A10. Similar ratios of activities were observed with extracts from HK293 and V79 cells (Table IGo). We also found catalytic activity with V79 expressed UGT1A3 (Table IGo), similar to preparations of UGT1A3 expressed in HK293 (26). In addition, expressed UGT1A1 was also active. Our results are somewhat different than Nowell et al. (26). It was reported that UGT1A8 was most active for the formation of N-hydroxy-PhIP-N2-glucuronide with no detectable activity with expressed UGT1A1. The preparation of UGT1A8 used in our experiments contained limited catalytic activity, while UGT1A1 was the most active toward this substrate.

The results of these studies indicate that several of the human UGTs are capable of glucuronidating both N-hydroxy-2AAF and N-hydroxy-PhIP, with UGT1A9 and UGT1A7 appearing to carry out this dual process most efficiently. In the following experiments, we have thus elected to utilize expressed UGT1A9 to examine its ability to interfere with the mutagenic activity of N-hydroxy-2-AAF and N-hydroxy-PhIP.

Induction of umuC gene expression by 2-AAF and PhIP through CYP1A2 metabolism
A quantitative umuC induction assay was used as a measurement of bioactivation of the aromatic amines. In control experiments, N-hydroxy-2AAF and N-hydroxy-PhIP demonstrated in a dose-dependent manner the ability to facilitate the mutagenic response as determined by an increase in S.typhimurium NM2009 ß-galactosidase activity (Figures 3A and 4AGoGo). In comparison, a step wise increase in ß-galactosidase activity was not observed with 2AAF or PhIP, which are both considered to be non-mutagenic in the absence of N-hydroxylation (Figures 3B and 4BGoGo). Thus, the N-hydroxylated metabolites serve as efficient candidates for analysis of mutagenic potential.



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Fig. 3. The genotoxicity of 2-AAF in Salmonella typhimurium NM2009. (a) This figure shows the umuC gene expression with indicated concentrations of N-hydroxy-2AAF. DMSO (10 µl) was used as a control. (b) In this figure, umuC gene expression was assessed by ß-galactosidase activity in tester strain NM2009 with indicated concentrations of 2-AAF, as described in Materials and methods. Group A (solid bars) contained human CYP1A2/NADHP reductase-containing membranes with a NADPH generating system. Group B (shaded bars) represents only 2-AAF. Group C was the same as group A but with 1 µM of the CYP inhibitor {alpha}-naphthoflavone.

 


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Fig. 4. The genotoxicity of PhIP in Salmonella typhimurium NM2009. (a) Dose–response of umuC gene expression with indicated concentrations of N-hydroxy-PhIP. (b) umuC gene expression analyzed by measuring ß-galactosidase activity in tester strain NM2009 with the indicated concentrations of PhIP. Group A included PhIP with the bioactivating components that contained E.coli membranes containing CYP1A2/NADPH reductase and the NADPH generating system (solid bars). Group B contained PhIP only (shaded bars).

 
To examine the ability of CYP1A2 to catalytically generate the mutagenic metabolites of 2AAF and PhIP as reported previously, the substrates were incubated with an NADPH generating system in the presence of membranes containing expressed CYP1A2/NADPH reductase. With both 2AAF and PhIP, a concentration-dependent increase in the mutagenic potential of CYP1A2 generated metabolites was observed (Figures 3B and 4BGoGo). The induction of ß-galactosidase activity in the presence of 2AAF was also inhibited by 1 µM of the CYP1A2 inhibitor, {alpha}-naphthoflavone (Figure 3BGo). Based upon the specificity of the oxidation reaction from previous experiments (13), the metabolites are generated through N-hydroxylation.

Inhibition of N-hydroxy 2-AAF genotoxicity by UGT1A9
Since it has been confirmed that UGT1A9 is capable of glucuronidating both N-hydroxy-2AAF and N-hydroxy-PhIP, experiments were conducted to examine the ability of UGT1A9 to inhibit the genotoxicity of these substrates. Cell lysates containing expressed UGT were used in a genotoxicity assay in S.typhimurium NM2009. In the presence of UDPGLcA, UGT1A9 inhibited the genotoxicity of N-hydroxy 2-AAF when incubated at a concentration of 25 µM and completely abolished genotoxicity at lower concentrations (Figure 5Go). In the absence of UDPGLcA or active UGT1A9 extract, there was no inhibition of mutagenicity. When incubations were performed with 2AAF as substrate in the presence of active CYP1A2 and UGT1A9, a similar profile leading to the inhibition of mutagenesis was observed (data not shown). Thus, the mutagenicity of N-hydroxy-2AAF can be blocked by active UGT1A9.



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Fig. 5. Effect of UGT1A9 metabolism on cells exposed to N-hydroxy-2AAF. Tester cells NM2009 were exposed to the indicated concentrations of N-hydroxy-2AAF in the presence of UGT1A9 containing cell lysates, as outlined in Materials and methods. The results were expressed as ß-galactosidase activity, which is an indicator of genotoxic activity.

 
Contrary to the glucuronidation of N-hydroxy 2-AAF, UGT1A9 was not able to interfere with the mutagenicity of N-hydroxy-PhIP (data not shown). This may be due to the dramatic differences in the formation of UGT1A9 generated glucuronide, as calculated from standard catalytic analysis (N-hydroxy-2AAF is 411 pmol/min/mg protein compared with 56.5 pmol/min/mg protein for N-hydroxy-PhIP). Since the mutagenic metabolites of N-hydroxy-PhIP are O-esterified acetyl derivatives formed from bacterial acetyltransferase, it is also possible that the efficiency of this reaction far exceeds that of glucuronidation, making it difficult to generate sufficient quantities of the glucuronide such that a reduction in mutagenicity can be elicited.


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CYP1A2 catalyzed N-hydroxylation of several classes of arylamines leads to the formation of metabolites that have been shown to initiate a mutagenic event. Interestingly, many of the mutagens, such as the dietary heterocyclic amines that are formed by N-hydroxylation, have also been detected as glucuronides. Since glucuronidation provides a means for inactivating the biological activity of drugs and xenobiotics, the formation of N-hydroxy-linked arylamine glucuronides would be expected to reduce the mutagenic activity of the CYP1A2 catalyzed N-hydroxy compounds. To investigate this possibility, two classical bacterial mutagens, 2AAF and PhIP, were chosen to examine the potential for human UDP-glucuronosyltransferases to interrupt the ability of these agents to promote a mutagenic response.

Both 2AAF and PhIP are benign substrates when added to a bacterial mutagenic assay, but their N-hydroxylated metabolites efficiently induce in a dose-dependent manner bacterial mutagenesis using the umuC test in S.typimurium TA1535/pSK1002, as developed previously by Oda et al. (32). When 2AAF and PhIP are used as substrates and incubated with E.coli membranes containing expressed CYP1A2 and the extracts added to the umuC test, a similar concentration dependent increase in bacterial mutagenesis is observed. Since the biological activity of the mutagens results in the formation of an N-acetoxy metabolite catalyzed by bacterial O-acetyl transferases and the interaction of the nitrenium ion with DNA, the relationship between the concentrations of the substrate used in the CYP1A2 reaction and the production of ß-galactosidase activity is a good indicator of the relative concentration of N-hydroxy metabolite. In these experiments, similar concentrations of 2AAF and PhIP lead to comparable mutagenic activity as catalyzed by CYP1A2. While N-hydroxy metabolite concentrations were not determined for 2AAF and PhIP, it can be concluded based upon the dose–response curves that similar levels of the N-hydroxy metabolites are needed to produce comparable mutagenic activity.

While considerable data are available on the ability of the different human UGTs to metabolize endogenous substances as well as xenobiotics and drugs (1), limited information is available on the ability of these enzymes to metabolize N-hydroxy metabolites. Our laboratory (21) has recently demonstrated that human UGT1A9 and UGT1A7 are capable of utilizing N-hydroxy-PhIP as substrate to form N-hydroxy glucuronides, but at relatively low rates in comparison to the aryl-O-linked phenolic glucuronidation of 7-hydroxybenzo[{alpha}]pyrene (21). It turns out that the glucuronidation of N-hydroxy-2AAF is fairly specific, serving as substrate primarily for UGT1A7 and UGT1A9 directed glucuronidation, although UGT1A3, UGT1A6, UGT1A8, UGT1A10 and UGT2B7 were capable of generating N-hydroxy-2AAF glucuronides but at dramatically reduced rates. The specificity of N-hydroxy-linked glucuronidation may be a favored reaction for UGT1A7 and UGT1A9, since these enzymes participate in the glucuronidation of N-hydroxy-2AAF and N-hydroxy-PhIP.

Although N-hydroxy-2AAF and N-hydroxy-PhIP exhibit comparable mutagenic potential and are glucuronidated by similar UGT isozymes, N-hydroxy-2AAF is conjugated far more efficiently by UGT1A9 than N-hydroxy-PhIP. The lack of comparable levels of glucuronidation is also reflected in the ability of UGT1A9 to prevent the N-hydroxy metabolites from initiating a mutagenic event in NM2009 cells. Addition of comparable concentrations of the N-hydroxy metabolites to the umuC test in the presence of active UGT1A9 led to dramatic reductions in N-hydroxy-2AAF initiated mutagenicity, while little effect was observed when similar experiments were carried out in the presence of N-hydroxy-PhIP. The ability of UGT1A9 to attenuate the mutagenic response of N-hydroxy-2AAF could also be demonstrated in a dose–response fashion using variable concentrations of substrate, indicating that glucuronidation can be rate limiting in uncoupling the mutagenic actions of this compound. When incubations are carried out with mixtures of active CYP1A2 and UGT1A9 in the presence of the pre-mutagens 2AAF and PhIP, UGT1A9 prevents CYP1A2 from generating sufficient unconjugated N-hydroxy-2AAF, as measured by the lack of SOS response in S.typhimuruim NM2009. However, UGT1A9 is not capable of disrupting the mutagenic actions of CYP1A2 directed N-hydroxylation of PhIP. We can suggest that while glucuronidation plays a role in the metabolism of N-hydroxy-PhIP, the efficiency for this reaction is not sufficient under saturating enzymatic conditions in the presence of CYP1A2 to detoxify the biologically active mutagen. Thus, bioactivation via CYP1A2 might be a more dominant pathway than glucuronidation initiated detoxification in overall PhIP metabolism.

CYP1A2 is expressed primarily in the liver and is the most efficient of the P450s in catalyzing the N-hydroxylation of the dietary heterocyclic amines such as PhIP and IQ (51). In rodents, it has been demonstrated that PhIP can lead to the formation of colon cancer and it is speculated that the N-hydroxy-PhIP produced in the liver may be the mutagen responsible for colon-initiated carcinogenesis. This experiment would suggest that CYP1A2 generated N-hydroxylation of PhIP does not serve as an efficient substrate for glucuronidation in vivo. The inefficiency of the human UGTs to trap N-hydroxy-PhIP appears to parallel this observation, suggesting that sufficient hydroxylated PhIP enters the systemic circulation avoiding additional metabolism. In addition, it has recently been demonstrated that while human colon expresses comparable levels of UGT1A9 RNA in addition to UGT1A1, UGT1A3 and UGT1A10, which are also capable of N-hydroxy-PhIP glucuronidation, rates of phenolic glucuronidation in human colon are 100-fold lower in UGT activity than that found in human liver (52). The lack of catalytic activity in colon is not explained by RNA abundance or protein accumulation, since both are comparable with those found in liver. If glucuronidation has evolved in human tissues to facilitate the detoxification of foreign chemicals, the colon is unable to participate in this process with the efficiency seen in other tissues, such as liver and intestine. Thus, if potential dietary carcinogens with potential to serve as substrates for glucuronidation are not efficiently metabolized in liver, the colon will not add substantially to the metabolism and detoxification of those agents.

The metabolism of potential carcinogens as studied in vitro remains to be examined in a complete functional biological system, where it might be possible to address issues relating to in vivo cellular carcinogen concentrations in relation to enzyme kinetic parameters, levels of activating and/or detoxifying enzyme expression and transportation of reactive intermediates to target organs. Yet the bacterial umuC test, which is an excellent model system to examine the potential mutagenic actions of heterocyclic and aromatic amines (32,53,54), has been exploited to study the potential for UGTs to influence the outcome of a patterned biological response such as mutagenesis. Coordination between CYP mediated bioactivation and UGT initiated detoxification has been demonstrated, with the glucuronidation of CYP1A2 activated N-hydroxy-2AAF leading to the interruption of the mutagenic response. By measuring a defined biological end point such as mutagenesis, this type of experiment allows for the alteration of defined variables such as protein and substrate concentration, and within a single measurement, makes it possible to predict the rate limiting step as controlled through bioactivation or detoxification of potential mutagens. In the presence of toxicants like N-hydroxy-2AAF, glucuronidation is capable of providing sufficient protection to limit the hazardous effects of the metabolite in initiating a mutagenic response.

This is in sharp contrast to the food derived heterocylic amines. Although PhIP has been shown to be a substrate for human glucuronidation, oxidative metabolism initiated by CYP1A2 leads to the formation of metabolites that are predisposed to initiate a mutagenic response while serving as substrates for glucuronidation. While analysis of N-hydroxy-PhIP has shown it to be a substrate for glucuronidation, UGT1A9 is limited catalytically in protecting the cell from the hazardous effects of the mutagen. However, care must be taken in the interpretation of these results, since mutagenic analysis as carried out in these experiments does not mimic an in vivo situation as might be encountered in the cell where a battery of UGT1A proteins may work in concert to glucuronidate these substrates. While these results are certainly preliminary, it would appear the glucuronidation may not be sufficient to prevent the potential hazardous effects of these compounds on the cell.


    Notes
 
4 To whom correspondence should be addressed Email: rtukey{at}ucsd.edu Back


    Acknowledgments
 
This work was conducted in part by support from US Public Health Service Grants CA79834 (R.H.T), R35 CA44353 and P30 ES00167 (F.P.G) and DFG 493/3-1 (C.P.S). The authors would also like to thank Deirdre Beaton for excellent technical assistance.


    Reference
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Reference
 

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Received November 9, 2000; revised February 13, 2001; accepted February 22, 2001.