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Research ArticleArticle

Glucuronidation of the Lung Carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) by Rat UDP-Glucuronosyltransferase 2B1

Qing Ren, Sharon E. Murphy, Andrew J. Dannenberg, Jong Y. Park, Thomas R. Tephly and Philip Lazarus
Drug Metabolism and Disposition September 1999, 27 (9) 1010-1016;

Abstract

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone and its major metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), are potent lung carcinogens in animals. UDP-glucuronosyltransferase (UGT)-mediated glucuronidation of NNAL is a potentially important detoxification pathway for these carcinogens. To identify the UGT isozyme(s) involved in this pathway, we examined the glucuronidation of NNAL in rat liver microsomes and homogenates from cell lines overexpressing specific UGT isozymes. NNAL glucuronidation was induced in liver microsomes from rats treated with family 2 UGT inducers including phenobarbitol and 3,5-di-tert-butyl-4-hydroxytoluene, which exhibited 1.7- and 2.6-fold higher rates of glucuronidation than microsomes from control rats. The rates of NNAL glucuronidation in liver microsomes from GUNN (deficient in family 1 UGTs) and RHA parental control rats were similar. All rat liver microsomes used in the present study catalyzed the glucuronidation of (S)-NNAL at a rate between 3.5 and 5.5 times that of the glucuronidation of (R)-NNAL. Liver microsomes from Wistar rats exhibiting the low-androsterone glucuronidation phenotype characteristic of the UGT2B2-deficient genotype glucuronidated NNAL at a rate similar to microsomes from Wistar rats exhibiting the high-androsterone glucuronidation phenotype/UGT2B2 [+] genotype. Homogenates from UGT2B1-overexpressing cells catalyzed the glucuronidation of NNAL at aKm of 745 μM. As with rat liver microsomes, NNAL-Gluc I was the major diastereomer formed by UGT2B1. Glucuronidation of NNAL was not detected with homogenates from UGT2B12-overexpressing cells. These results suggest that UGT2B1 plays an important role in the glucuronidation of NNAL in the rat.

One of the most abundant potent procarcinogens in tobacco and tobacco smoke is the nicotine-derived tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)2 (Hecht, 1998; Hecht and Hoffmann, 1989). In mainstream cigarette smoke, the total level of NNK is 3 to 15 times that of benzo[a]pyrene (Adams et al., 1987). NNK induces predominantly lung adenocarcinomas in rodents independent of the route of administration (reviewed in Hecht, 1998). In the Fischer 344 rat, NNK induces pancreatic tumors (Rivenson et al., 1988) and, when applied with the related tobacco-specific nitrosamine,N′-nitrosonornicotine, oral cavity tumors (Hecht et al., 1986). The cumulative dose of 1.8 mg NNK/kg body weight, which induces lung tumors in rats (Belinsky et al., 1990), is similar to the cumulative lifetime dose of 1.6 mg NNK/kg body weight estimated for the average 2-pack-a-day U.S. smoker (Hecht, 1989; Hecht and Hoffmann, 1989). NNK is therefore considered to be a likely causative agent for cancers of the lung, oral cavity, and pancreas in humans (Hecht and Hoffmann, 1989).

The major metabolic pathway for NNK in most tissues is conversion to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) by carbonyl reduction (see Fig. 1A). NNK reduction to NNAL occurs in rodents, monkeys, and humans (Carmella et al., 1993; Hecht et al., 1993; Hecht, 1998), with an estimated 39 to 100% of the NNK dose converted to NNAL in smokers (Carmella et al., 1998). NNAL is activated via pathways similar to those observed for NNK and, like NNK, is a potent lung and pancreatic carcinogen in rodents (Rivenson et al., 1988;Hecht, 1998). In a reaction likely to be catalyzed by the UDP-glucuronosyltransferase (UGT) family of enzymes, NNAL is also metabolized to its glucuronide, β-O-[4-(methylnitrosamino)-1-(3-pyridyl)-1-but-1-yl]-d-glucosiduronic acid (NNAL-Gluc; see Fig. 1A; Morse et al., 1990; Carmella et al., 1993; Hecht et al., 1993; Hecht, 1998). Because NNK is metabolized to both the (S) and (R) enantiomers ofNNAL, two diastereomeric glucuronides of NNAL are formed (see Fig. 1B). These have previously been referred to as NNAL-Gluc I, formed from (S)-NNAL, and NNAL-Gluc II, formed from (R)-NNAL (Hecht et al., 1997). NNK-treated rats form predominantly NNAL-Gluc I (Hecht et al., 1997; Murphy et al., 1997) due primarily to the preferential glucuronidaton of (S)-NNAL rather than preferential reduction of NNK to (S)-NNAL. In a recent study, rat liver microsomes were shown to metabolize racemic NNAL to NNAL-Gluc I (Staretz et al., 1997). Therefore, in the rat, the predominance of NNAL-Gluc I appears to be due to the preferential glucuronidation of (S)-NNAL.

Figure 1
Figure 1

Simplified schematic of NNK metabolism to NNAL-Gluc (A) and structures of NNAL and NNAL-Gluc rotamers and enantiomers (B).

Both NNAL and NNAL-Gluc are excreted in the bile of rats (Shulze et al., 1992) and in the urine of animals, smokers, and individuals exposed to sidestream smoke (Morse et al., 1990; Hecht et al., 1994; Murphy et al., 1994; Carmella et al., 1995; Richie et al., 1997; Parsons et al., 1998). Although the formation of NNALis clearly not a detoxification pathway for NNK, the glucuronidation ofNNAL appears to be an important mechanism for NNK detoxification. Recent studies have demonstrated that, in contrast to the high tumorigenicity exhibited by both (R)- and (S)-NNAL, NNAL-Gluc is nontumorigenic after s.c. injection into A/J mice (Upadhyaya et al., 1999). In addition, the glucuronidation of (S)-NNAL was significantly greater than (R)-NNAL after injection into A/J mice, a pattern consistent with the higher tumorigenicity exhibited by (R)-NNAL in the same experiments (Upadhyaya et al., 1999). Kim and Wells (1996) also reported that skin fibroblasts from UGT family 1-deficient rats were more sensitive to NNK-mediated cytotoxicity. Significant interindividual variability in the ratio of NNAL-Gluc:NNAL excreted in human urine has been observed, suggesting that individuals may differ greatly in their ability to detoxify NNK (Carmella et al., 1995). It has been hypothesized that the ability of an individual to glucuronidate NNAL may directly affect their susceptibility to lung and potentially other tobacco-related cancers.

UGTs are a superfamily of enzymes that glucuronidates many xenobiotics and endogenous compounds (Tephly and Burchell, 1990). Based on differences in sequence homology and substrate specificity, two families of UGTs (UGT1 and UGT2) have been identified in several species, each containing several highly homologous UGT genes. We have shown previously that the human family 1 isozyme UGT1A9 (for updated nomenclature, see Mackenzie et al., 1997) catalyzed the glucuronidation of NNAL; no activity was observed with the related human UGT family 1 isozyme UGT1A6 (Ren et al., 1996). In the rat, phenobarbitol (PB), which has been associated with the induction of family 2 UGT isozymes in the rat (Mackenzie, 1986; Pritchard et al., 1994; Emi et al., 1995; Green et al., 1995), induces the glucuronidation of two NNK metabolites, 4-(hydroxymethylnitrosamino)-1-(3-pyridyl)-1-butanone and 4-hydroxy-1-(3-pyridyl)-1-butanone (Murphy et al., 1994, 1997). The goal of the present study was to elucidate the UGT(s) responsible for the glucuronidation of NNAL. We demonstrate that theNNAL glucuronidation pathway is specific to UGT family 2 isozymes in rats and that one of the major isozymes involved in this metabolic pathway is UGT2B1.

Materials and Methods

Chemicals and Materials.

PB, 3-methylcholanthrene (3-MC), clofibric acid, 2[3]-tert-butyl-4-hydroxyanisole (BHA), 3,5-di-tert-butyl-4-hydroxytoluene (BHT), 4-nitrophenol (4-NP), androsterone, UDP-glucuronic acid (UDPGA),d,l-2-lysophosphatidyl choline palmital C16:0, and β-glucuronidase were purchased from Sigma (St. Louis, MO). Bilirubin was obtained from Fluka Chemicals (Ronkonkoma, NY). [14C]UDPGA (specific activity = 318 Ci/mol) was obtained from ICN Pharmaceuticals (Costa Mesa, CA), and [3H]NNK (specific activity = 2.4 Ci/mmol) as well as unlabeled NNK were purchased from Chemsyn Scientific (Lenexa, KS). Dulbecco’s modified Eagle’s medium was obtained from Mediatech (Herndon, VA), whereas fetal bovine serum and geneticin were purchased from GIBCO BRL Life Technologies (Grand Island, NY). Internal standards for NNAL glucuronidation studies, including 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB), 4-(methylnitrosamino)-1-(N-oxy-3-pyridyl)-1-butanone (NNK-N-oxide), 4-(methylnitrosamino)-1-(N-oxy-3-pyridyl)-1-butanol (NNAL-N-oxide), 4-hydroxy-1-(3-pyridyl)-1-butanol (diol), and 4-oxo-4-(3-pyridyl)-butyric acid (keto acid), were kind gifts from Shantu Amin at the American Health Foundation (Valhalla, NY).

Animal Studies.

Sprague-Dawley (SD) rats (150–200 g, female), GUNN rats (j/j; 150–200 g, female), and RHA rats (+/+; 150–200 g, female) were purchased from Harlan Sprague-Dawley, Inc. (Indianapolis, IN). SD rats (n = 3–5 per group) were treated with one of the following: PB (40 mg · kg body weight−1 · day−1 for 4 days), clofibric acid (250 mg · kg body weight−1 · day−1 for 3 days), 3-MC (40 mg · kg body weight−1 · day−1 for 4 days) or vehicle (corn oil or water) by i.p. injection and fed rat chow (NIH07) ad libitum. GUNN and RHA rats were untreated and were fed rat chow ad libitum. The purchase and treatment of Wistar rats with BHA (0.75%, w/w) or BHT (0.5%, w/w) has been described (Kashfi et al., 1994). Livers (10–15 g) were excised from rats sacrificed by exposure to carbon dioxide (CO2), and rat liver microsomes were prepared through differential centrifugation as described (Coughtrie et al., 1987). Rat liver microsomes (10–20 mg protein/ml) were stored at −70°C in 100-μl aliquots, with total protein concentrations measured using the BCA assay (Pierce Corp., Rockford, Il).

Cell Homogenates.

HK293 (human kidney fibroblast) cells and HK293 cell lines overexpressing UGT2B1 or UGT2B12 (Green et al., 1995; King et al., 1997) were grown to 80% confluence in Dulbecco’s modified Eagle’s medium supplemented with 4.5 mM glucose, 10 mM HEPES, 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin and maintained in 700 μg/ml geneticin for selection of UGT overexpression, in a humidified incubator in an atmosphere of 5% CO2. Cells were suspended in Tris-buffered saline (25 mM Tris base/138 mM NaCl/2.7 mM KCl, pH 7.4) and subjected to three rounds of freeze-thaw before gentle homogenization. Cell homogenates (5–30 mg/ml) were stored at −70°C in 100-μl aliquots. Total cell homogenate protein concentrations were determined using the BCA assay as described above.

NNAL Glucuronidation Assays.

The rate of NNAL glucuronidation by rat liver microsomes was determined using the following conditions. Microsomal protein (50 μg) was incubated in 50 mM Tris-HCl (pH 7.5)/10 mM MgCl2 (100 μl volume) containing 10 μgd,l-2-lysophosphatidyl choline palmital C16:0 with 4 mM UDPGA and 1 mM [3H]NNAL(0.5 μCi) at 37°C for 40 min. (The rate of NNAL-Gluc formation was linear for at least 40 min for all rat liver microsomal samples tested.) Reactions were terminated by the addition of 1:10 vol of 0.3 N Ba(OH)2/0.3 N ZnSO4 on ice. The precipitate was removed by centrifugation, and the supernatant was filtered and analyzed for glucuronidated NNAL metabolites by HPLC with radioflow detection as described (Murphy et al., 1995). A Waters Associates dual-pump (model 510) HPLC system (Milford, MA), equipped with an automatic injector (WISP model 710B), a UV detector operated at 254 nm (model 440), and a Beta radioactive flow detector (INUS Systems, Fairfield, NJ) was used. NNAL metabolites were separated on a 5 μm partisphere C18 column (4.6 mm × 250 mm Whatman, Clifton NJ) as described (Murphy et al., 1995). NNK metabolite standards (3–10 μg, see chemicals above) were coinjected with the reaction mixture and monitored by UV detection (254 nm). [3H]NNAL-Gluc peaks were tentatively identified by relative retention time, then confirmed by sensitivity toEscherichia coli β-glucuronidase treatment. Fractions containing [3H]NNAL-Gluc were treated with 1,000 U of E. coli β-glucuronidase at 37°C for 12 to 16 h and analyzed for released NNAL by radioflow-HPLC as described above.

The glucuronidation of NNAL by UGT2B1- and UGT2B12-overexpressing as well as non-UGT-expressing HK293 cell lines was analyzed using the following conditions. Cell homogenate protein (0.25–5.0 mg) was incubated at 37°C in 50 mM Tris-HCl (pH 7.5)/10 mM MgCl2 (100–800 μl volume) containing 0.1 μg/μl d,l-2-lysophosphatidyl choline palmital C16:0, 2 mM [14C]UDPGA (0.5–10 μCi, depending on reaction volume) and 0.5 mM NNAL. [14C]Glucuronidated products were analyzed by radioflow HPLC as described above or by thin-layer chromatography (TLC) and autoradiography (Bansal and Gessner, 1980; described below). [14C]NNAL-Gluc peaks were tentatively identified by relative retention time, and confirmed by sensitivity toE. coli β-glucuronidase added to the assay mixture as described above.

Aglycone Glucuronidation Assays.

UGT activity toward 4-NP in rat liver microsomes was determined using the method of Burchell and Weatherill (1981). Enzyme reactions (125 μl final volume) containing 250 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 4 mM UDPGA, 5 μg ofd,l-2-lysophosphatidyl choline palmital C16:0, 25 μg of protein, and 0.5 mM 4-NP were incubated at 37°C for 5 min. [The rate of 4-nitrophenol-O-glucuronide (4-NP-gluc) formation was linear for up to 10 min for all rat liver microsomal samples tested.] Reactions were terminated by the addition of 2 vol of 0.5 M ice-cold trichloroacetic acid and centrifuged at 17,000g in a microcentrifuge for 5 min. The supernatants were collected and analyzed by spectrophotometry at 400 nm for loss of absorbance to determine the conversion of 4-NP to its glucuronide. The presence of 4-NP-gluc was confirmed by sensitivity to treatment toE. coli β-glucuronidase as described above.

The glucuronidation of 1) 4-NP in cell homogenates and 2) other aglycones in both rat liver microsomes and in cell homogenates was determined by TLC analysis as described by Bansal and Gessner (1980). Enzyme assay mixtures (100 μl final volume) of 50 mM Tris-HCl (pH 7.5)/10 mM MgCl containing 10 μg ofd,l-2-lysophosphatidyl choline palmital C16:0, 250 μg (cell homogenate) to 500 μg (rat liver microsomes) protein, 1–2 mM [14C]UDPGA (0.1–0.5 μCi/reaction), and 12 to 1680 μM aglycone were incubated at 37°C for up to 40 min. Reactions were terminated by the addition of 2 vol of ice-cold 100% ethanol, then centrifuged at 13,000 rpm in a microcentrifuge. Supernatants were collected, dried, and resuspended in 10 to 20 μl of water. Aliquots were applied to TLC plates and developed in organic solvent as described (Bansal and Gessner, 1980). [14C]4-NP-gluc, previously purified by HPLC, was used as a TLC reference. The chromatograms were air-dried and exposed for autoradiography for 1 to 2 weeks. Quantification was performed by densitometric readings of autoradiographs using computer analysis (Photoshop, National Institutes of Health Image 1.61 analysis system for Macintosh). As with other glucuronidation analysis, glucuronide formation was confirmed by treatment with E. coli β-glucuronidase as described above.

Statistical Analysis.

The Student’s t test (two-tailed) was used for all comparative analyses. The correlation coefficient (r2) from linear regression analysis of Lineweaver-Burk plots was used to determine theKm and Vmax of UGT2B1 glucuronidation of NNAL.

Results

NNAL Glucuronidation in Liver Microsomes from Induced Rats.

Rat UGT isozymes are responsive to various enzyme inducers (Emi et al., 1995). For example, clofibric acid and 3-MC were previously shown to be effective inducers of the bilirubin- and phenol-metabolizing family 1 UGTs, respectively (Emi et al., 1995). BHT and PB have been previously associated with the induction of family 2 UGTs (Mackenzie, 1986; Pritchard et al., 1994; Emi et al., 1995; Green et al., 1995; Yang et al., 1996), whereas BHA was shown to induce the glucuronidation of small, planar phenols (Kashfi et al., 1994; Yang et al., 1996). To examine whether the glucuronidation of NNALis susceptible to these various inducers, the rate of NNALglucuronidation was determined in liver microsomes prepared from untreated as well as PB-, 3-MC-, or clofibric acid-treated SD rats. In addition, the levels of glucuronidated NNAL were examined in liver microsomes prepared previously from untreated as well as BHA- or BHT-treated Wistar rats (Kashfi et al., 1994). In vitro assays demonstrated that significant levels of induction of NNALglucuronidation were observed in liver microsomes from PB- (p < .05), BHA- (p < .05), and BHT- (p < .01) treated rats as compared to liver microsomes from corresponding untreated controls (Table 1). Significant levels of induction were not observed in liver microsomes from either 3-MC- (p = .40) or clofibric acid- (p = .10) treated rats. A similar pattern was observed with PB, 3-MC, and clofibric acid in Fischer 344 rats (results not shown). This pattern of induction is consistent with the possibility that NNAL glucuronidation is preferentially mediated by family 2 UGT isozymes.

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Table 1

Induction of glucuronidation in rat liver microsomes

4-NP is glucuronidated by multiple UGT family 1 and family 2 isozymes (Tephly and Burchell, 1990). As a control for the induction of UGT activity, the rate of 4-NP-gluc formation was shown to be significantly induced in liver microsomes from 3-MC- (p < .05), PB- (p < .05), BHA- (p < .005), and BHT- (p < .001) induced rats, but not in microsomes from clofibric acid-treated rats (Table 1). These levels of induction were similar to those reported previously for liver microsomes from rats treated with these agents (Falany and Tephly, 1983; Pritchard et al., 1994; Ikushiro et al., 1995). As observed in previous studies (Ikushiro et al., 1995), significant levels of induction of bilirubin glucuronidation were observed in microsomes from clofibric acid-treated rats (p < .05; Table1).

As illustrated in Fig. 2 for control and PB-induced microsomes (B and C), the glucuronides of (R)- and (S)-NNAL separate under the HPLC conditions of our analysis. As described in previous studies (Murphy et al., 1995), NNAL-Gluc peaks eluted at a retention time between twoN-oxidation metabolites of NNAL and NNK, NNAL-N-oxide and NNK-N-oxide (between 31 and 38 min). The pair of early eluting peaks (peaks 1 and 2) appear to correspond to the E and Z isomers of NNAL-Gluc I, with peaks 3 and 4 to the E and Z isomers of NNAL-Gluc II (Hecht et al., 1997; see Fig. 1). These peaks were observed using either [3H]NNAL (Fig.2) or [14C]UDPGA (results not shown) as the radiolabeled substrate. When this entire region was collected and treated with β-glucuronidase, the only product was NNAL(Fig. 2D), confirming that these peaks correspond to glucuronidatedNNAL conjugates. All rat liver microsomes analyzed, whether induced or not, preferentially catalyzed the glucuronidation of (S)-NNAL (i.e., the formation of NNAL-Gluc I; Table 1). The ratio of NNAL-Gluc I to NNAL-Gluc II formed in liver microsomes from control and induced SD rats was not significantly different and averaged approximately 4.5:1 (Table 1). A similar ratio was observed in liver microsomes from control as well as PB-induced Fischer 344 rats (results not shown). The ratio obtained using liver microsomes from control Wistar rats was lower and was significantly increased by BHT (p < .01) and BHA treatment (p < .05; Table 1).

Figure 2
Figure 2

HPLC analysis of NNAL-Gluc formation by liver microsomes from untreated and PB-treated SD rats.

Liver microsomes (50 μg of protein) from SD rats were incubated with 1 mM [3H]NNAL (0.5 μCi) and 4 mM unlabeled UDPGA in a 100-μl reaction volume as described in Materials and Methods. A, NNK metabolite standards (3–10 μg each metabolite). B, [3H]NNAL metabolites formed by liver microsomes from untreated control rats. C, [3H]NNAL metabolites formed by liver microsomes from PB-treated rats (40 mg · kg body weight−1 · day for 4 days). D, analysis of the β-glucuronidase-treated [3H]NNAL-Gluc fraction (30–37 min, from liver microsomes from PB-treated rats; details inMaterials and Methods).

NNAL Glucuronidation in Liver Microsomes from GUNN Rats.

GUNN (j/j) rats are deficient in family 1 UGTs due to the presence of a homozygous 1-bp deletion within the common region of the UGT family 1 locus (i.e., position +1239; Iyanagi, 1991). Therefore, to analyze the role of family 1 UGTs in the glucuronidation of NNAL, the rate of NNAL glucuronidation in liver microsomes from GUNN rats was determined and compared to the rate observed in liver microsomes from parental RHA (+/+) controls. As shown in Table2, the rate of NNALglucuronidation was similar for liver microsomes from both strains of rat. In contrast, the rate of 4-NP glucuronidation, which is catalyzed by the phenol-metabolizing family 1 UGTs as well as other UGT isozymes (Tephly and Burchell, 1990; Ikushiro et al., 1995), was approximately 3-fold lower in liver microsomes from GUNN (j/j) rats (p < .025) as compared with RHA (+/+) controls (Table 2). Therefore, the family 1 UGTs, which are deficient in the GUNN rat, do not appear to play a role in the glucuronidation ofNNAL. These data are consistent with the lack of significant induction of NNAL glucuronidation observed in liver microsomes from rats treated with either 3-MC or clofibric acid, agents that induce mainly family 1 UGTs (Ikushiro et al., 1995).

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Table 2

Glucuronidation in liver microsomes from GUNN (j/j; UGT family 1-deficient) and RHA (+/+) control rats

NNAL Glucuronidation in Liver Microsomes from Untreated Wistar Rats.

Our results with inducers and the GUNN rat suggest that NNALglucuronidation may be mediated by family 2 UGTs. Thirty-fifty percent of Wistar rats exhibit a homozygous gene deletion of the family 2 isozyme, UGT2B2 (Corser et al., 1987; Homma et al., 1992). Androsterone is largely glucuronidated by UGT2B2, and this corresponds with an observed bimodal pattern of androsterone glucuronidation in Wistar rats [i.e., UGT2B2-deleted rats exhibit a low androsterone-glucuronidating phenotype (Matsui et al., 1979; Green et al., 1985; Corser et al., 1987; Homma et al., 1992)]. Therefore, to investigate the role of UGT2B2 in NNAL glucuronidation, we compared the relative rates of NNAL glucuronidation to androsterone glucuronidation in liver microsomes from untreated Wistar rats. Microsomes from three of five (60%) untreated Wistar rats examined in this study exhibited the low androsterone-glucuronidating phenotype typical of UGT2B2-deficient rats (Table3). However, no correlation was observed between the levels of glucuronidated NNAL and androsterone phenotype. The same lack of correlation between NNALglucuronidation and androsterone glucuronidation was also observed in liver microsomes from BHA- (n = 5) and BHT- (n = 5) treated Wistar rats (results not shown). These data suggest that UGT2B2 does not play a major role in the glucuronidation of NNAL.

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Table 3

Comparison of NNAL- and androsterone-glucuronidating activities in liver microsomes from untreated Wistar rats

NNAL Glucuronidation in Cell Homogenates from UGT Family 2 Isozyme-Overexpressing Cell Lines.

To further investigate the role of family 2 UGTs in the glucuronidation of NNAL, we examined the rate of NNALglucuronidation by cellular homogenates prepared from HK293 cells that overexpressed either UGT2B1 or UGT2B12 (Green et al., 1995; King et al., 1997). The NNAL glucuronidation activities of the two UGT-overexpressing cell lines was determined and compared with the activity with previously characterized substrates (Table4). The relative glucuronidation activity of UGT2B1 was determined using clofibric acid as the aglycone, whereas 4-NP was the aglycone used to assay for UGT2B12 activity. The glucuronidated conjugates of both aglycones were sensitive to β-glucuronidase treatment. Using these two substrates, the glucuronidating activity of homogenates prepared from UGT2B1-overexpressing cells was approximately 15.5-fold that of homogenates prepared from UGT2B12-overexpressing cells (Table 4). Therefore, to analyze presence of NNAL glucuronidating activity in the two cell lines, 15.5 times more homogenate protein was used for UGT2B12-overexpressing cells (i.e., 3.9 mg) than for UGT2B1-overexpressing cells (0.25 mg). No glucuronidation ofNNAL was observed by homogenates from cells overexpressing UGT2B12 (Fig. 3D), but significant levels of NNAL glucuronidation were observed in homogenates prepared from UGT2B1-overexpressing cells (Fig. 3B; Table 4). The14C-labeled product formed by homogenates from UGT2B1-overexpressing cells was sensitive to β-glucuronidase treatment (Fig. 3C). Based on their relative retention times when compared to: 1) the retention times of peaks 1 and 2 observed in rat liver microsomes (31–34 min; see Fig. 2) and 2) the retention times of NNK metabolites, the UGT2B1-catalyzed NNAL-Gluc peaks (shown in Fig.3C) have been identified as the two rotamers (i.e., E andZ; see Fig. 1) of the glucuronide of (S)-NNAL (NNAL-Gluc I). NNAL-Gluc I is the major diasteromer: 1) formed by rat liver microsomes (Table 1) and 2) excreted in the urine from NNK-treated rats (Hecht et al., 1997; Murphy et al, 1997). The Km for the glucuronidation of NNAL by UGT2B1 was 745 μM with aVmax of 27.5 pmol · mg protein−1 · min−1 as determined by Lineweaver-Burk kinetic analysis (Fig.4). Ther2 value from this analysis (r = 0.987) suggests that the enzyme kinetics of this reaction is linear at the range of NNAL concentrations used (0.25–2 mM).

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Table 4

Glucuronidating activities in UGT2B1- or UGT2B12-overexpressing HK293 cell lines

Figure 3
Figure 3

HPLC analysis of NNAL-Gluc formation in UGT-overexpressing HK293 cell lines.

Cell homogenates were incubated at 37°C for 40 min with 0.50 mMNNAL and 2 mM [14C]UDPGA as described inMaterials and Methods. A, NNK metabolite standards (3–10 μg each metabolite). B, 14C-labeled metabolites from incubations using cell homogenates (1 mg) from UGT2B1-overexpressing cells. C, 14C-labeled metabolites from incubations using cell homogenates (1 mg) from UGT2B1-overexpressing cells after subsequent incubation with β-glucuronidase (1000 U, 37°C, 60 min). D, 14C-labeled metabolites from incubations using cell homogenates (5 mg) from UGT2B12-overexpressing cells.

Figure 4
Figure 4

Lineweaver-Burk plot of NNALglucuronidation in UGT2B1-overexpressing cell homogenates.

Kinetic analysis of NNAL glucuronidation activity was performed in cell homogenates from the UGT2B1-overexpressing cell line by HPLC as described in Materials and Methods except that NNAL concentrations ranged from 0.25 to 2 mM. All incubations were performed for 5 min at 37°C (the rate ofNNAL glucuronidation was linear in UGT2B1-overexpressing for up to 10 min incubation).

Discussion

In this study, we present results of experiments designed to determine the UGT isozyme(s) that play a role in the glucuronidation ofNNAL in the rat. This is the first study to directly measure the glucuronidation of this potent lung carcinogen. Significant increases in the rate of NNAL glucuronidation were observed in liver microsomes prepared from rats treated with inducers of UGT family 2 isozymes. In contrast, liver microsomes prepared from rats treated with family 1 inducers did not catalyze NNALglucuronidation at a higher rate then did noninduced microsomes. These data are consistent with the fact that no alteration in the rate ofNNAL glucuronidation were observed in liver microsomes from rats deficient in UGT family 1 isozymes (i.e., in GUNN rats). Taken together, these data suggest that family 1 UGTs do not play a major role in the glucuronidation of NNAL. The fact that Kim and Wells (1996) demonstrated increased NNK-mediated cytotoxicity in skin fibroblasts from GUNN as compared to RHA control rats would suggest that, potentially, one or more UGT family 1 isozymes may be involved in the glucuronidation of other carcinogenically active NNK metabolites other than NNAL, such as 4-(hydroxymethylnitrosamino)-1-(3-pyridyl)-1-butanone. The high degree of specificity exhibited by UGT isozymes that appears to be exhibited toward NNK metabolites suggests that, potentially, multiple UGT isozymes from both UGT families may be important determinants in overall susceptibility to NNK-induced carcinogenicity.

The data presented in this study also provide information as to theNNAL-glucuronidating capacity of three family 2 rat UGT isozymes, UGT2B1, UGT2B2, and UGT2B12. Of these, only UGT2B1 appears to metabolize NNAL. The fact that homogenates of cell lines that overexpressed UGT2B1 catalyzed NNAL glucuronidation efficiently is consistent with the induction of NNALglucuronidation observed in liver microsomes from rats treated with PB or BHT, because both agents have been associated with the induction of this UGT isozyme (Mackenzie, 1986; Pritchard et al., 1994; Green et al., 1995; Yang et al., 1996). Additional support for the role of UGT2B1 in the metabolism of NNAL is provided by the stereoselectivity of this reaction. NNAL-Gluc I was the major NNAL-Gluc diasteromer formed in rat liver microsomes and in homogenates from UGT2B1-overexpressing cells. In addition, BHT, which has been previously demonstrated to be a strong inducer of UGT2B1 (Yang et al., 1996), exhibited a preferential and significant induction of NNAL-Gluc I.

UGT2B1 has a very wide substrate specificity and catalyzes the glucuronidation of acids such as clofibric acid, as well as phenols such as morphine. The Km of NNALglucuronidation by UGT2B1 (745 μM) was intermediate to other substrates studied, where the Km ranged from 12 μM for clofibric acid to 3.18 mM for morphine (Pritchard et al., 1994) and was similar to the Km of 432 μM reported for nalorphine (King et al., 1997). UGT2B1 has also been shown to possess a high degree of stereoselectivity. Pritchard and coworkers (1994) previously reported that UGT2B1 preferentially glucuronidated the (S)-enantiomer of ibuprofen, analogous to the preference we report here for (S)-NNAL. Because relatively high levels of UGT2B1 expression were previously observed in rat liver (Mackenzie, 1987), these data suggest that UGT2B1 plays a major role in the glucuronidation and elimination ofNNAL in the rat.

The major diastereomeric form of glucuronidated NNAL in urine from snuff users (Murphy et al., 1994) as well as in microsomes prepared from human livers (Ren et al., 1996) is NNAL-Gluc II. As described in the present study, the major diasteromeric form of NNAL-Gluc in rat liver microsomes is NNAL-Gluc I, a pattern consistent with that observed previously in urine from NNK-treated rats (Hecht et al., 1993). Although the present studies demonstrate a lack of significant activity exhibited by family 1 UGTs for NNAL in the rat, preliminary results from this laboratory demonstrated that the human family 1 UGT isozyme, UGT1A9, catalyzes the glucuronidation ofNNAL (Ren et al., 1996). Thus, there appears to be species-specific differences in terms of the UGT isozymes involved in the glucuronidation of NNAL in rats as compared with humans. UGT2B1 exhibits approximately 75% homology at the nucleotide level and 60% homology at the amino acid level with several human UGTs, with the highest homology existing with UGT2B15 (77% homology at the nucleotide level, 66% at the amino acid level; analysis performed by Blast search of National Center for Biotechnology Information sequence database, National Institutes of Health). Further studies examining known human UGT isozymes in the glucuronidation of NNAL are currently under way.

Acknowledgments

We thank Stephen Strudwick and James Liu, Department of Pathology and Laboratory Medicine, Temple University School of Medicine, for their helpful discussions.

Footnotes

  • Send reprint requests to: Philip Lazarus, Ph.D., Department of Pathology and Laboratory Medicine, OMS Bldg., Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140. E-mail: plazarus{at}vm.temple.edu

  • ↵1 This research was presented at the American Association for Cancer Research meeting, 1998, in New Orleans, LA. (Proc. Amer. Assoc. Cancer Res.)

  • This research was supported by National Institutes of Health Grants CA59917 (P.L.), DE12206 (P.L.), and CA73173 (J.Y.P.).

  • Abbreviations used are::
    NNK
    4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
    NNAL
    4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol
    UGT
    UDP-glucuronosyltransferase
    UGT
    UDP-glucuronosyltransferase
    NNAL-Gluc
    β-O-[4-(methylnitrosamino)-1-(3-pyridyl)-1-but-1-yl]-d-glucosiduronic acid
    PB
    phenobarbitol
    3-MC
    3-methylcholanthrene
    BHA
    2[3]-tert-butyl-4-hydroxyanisole
    BHT
    3,5-di-tert-butyl-4-hydroxytoluene
    4-NP
    4-nitrophenol
    UDPGA
    UDP-glucuronic acid
    SD
    Sprague-Dawley
    4-NP-gluc
    4-nitrophenyl-O-glucuronide
    TLC
    thin-layer chromatography
    • Received February 9, 1999.
    • Accepted April 26, 1999.

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Glucuronidation of the Lung Carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) by Rat UDP-Glucuronosyltransferase 2B1

Qing Ren, Sharon E. Murphy, Andrew J. Dannenberg, Jong Y. Park, Thomas R. Tephly and Philip Lazarus
Drug Metabolism and Disposition September 1, 1999, 27 (9) 1010-1016;
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