Chem. Res. Toxicol. 1996,8,772-779
772
Glucuronidation of 4-((Hydroxymethy1)nitrosamino)1-(3-pyridyl)-l-butanone, a Metabolically Activated Form of 4-(Methy1nitrosamino)1-(3-pyridy1)-1-butanone,by Phenobarbital-TreatedRats Sharon E. Murphy," Deborah A. Spina, Maria G . Nunes, and Dominic A. Pull0 American Health Foundation, Valhalla, New York 10595 Received November 10, 1994@
In the rat, 4-(methylnitrosamino)-l-(3-pyridyl)~l-butanone (NNK) induces lung tumors independent of the route of administration. To exert its carcinogenic potential, NNK must be metabolically activated. Like most nitrosamines NNK is activated by a-hydroxylation. The striking tissue specificity of tumor induction by nitrosamines has been primarily attributed to the efficient a-hydroxylation of a particular nitrosamine by its target tissue. Two other factors which may contribute to this are the following: the relative capacity of different tissues to detoxify the a-hydroxynitrosamine and the preferential uptake of the active metabolite by the target tissue. In the present study we report the characterization of the O-glucuronide of 4-((hydroxymethyl)nitrosamino)-l-(3-pyridyl)-l-butanone (a-hydroxymethylNNK-Gluc). The formation of this glucuronide could either serve a s a detoxification pathway or provide a stable transport form of the a-hydroxylated metabolite. I n addition, the metabolism of NNK to a glucuronide of the a-hydroxynitrosamine provides the first definitive evidence for the formation of a-hydroxymethylNNK. a-HydroxymethylNNK-Gluc was present in the urine of rats treated with phenobarbital (PB) and NNK. It was also formed by hepatocytes from PB-treated rats, accounting for 4% of the total metabolites in the media following incubation with 1pM NNK. The data that support the identity of this metabolite as a-hydroxymethylNNK-Gluc are as follows. (1)Incubation of this metabolite with /?-glucuronidase resulted in the quantitative release of 4-hydroxy-l-(3-pyridyl)-l-butanone (HPB), the decomposition product of a-hydroxymethylNNK. (2)This glucuronide was detected by radioflow HPLC analysis when NNK which was tritium labeled in either the pyridine ring or the methyl group was used. Therefore, it contains both these functional groups. (3)When hepatocyte media was analyzed for this glucuronide (molecular weight 399)using LC/MS with selected ion monitoring in the positive ion mode, a peak that was sensitive to /?-glucuronidase treatment, with mlz (M H), was detected a t the correct retention time. LC/MS/MS analysis of this peak with selection of mlz 400 generated daughter ions of mlz 206,176,148,and 106. This fragmentation is consistent with this metabolite being a-hydroxymethylNNK-Gluc.
+
ylene carbons adjacent to the N-nitroso group, is the pathway responsible for the activation of NNK to DNA The tobacco-specific nitrosamine 4-(methylnitrosamino)binding species (1,4-7; Figure 1). The presence of these 1-(3-pyridyl)-l-butanone (NNK)l induces lung tumors in pathways has been demonstrated in primates, the marrodents, independent of the route of administration, and moset and patas monkey, as well as in smokers (8-111. is believed to be a causative factor in human lung cancer In addition to a-hydroxylation, NNK may also be (1,Z). In rats, NNK also causes tumors of the liver, nasal metabolized by N-oxidation or carbonyl reduction (Figure cavity, and pancreas (3). To exert its carcinogenic 1). The enzymes responsible for all three of these potential NNK must be metabolically activated. Numerpathways are present in a number of different tissues. ous studies in rats and mice have established that In the rat, these include the liver, lung, esophagus, and a-hydroxylation of NNK, at either the methyl or methoral and nasal cavity tissue ( I , 12-17). It has been proposed that the sensitivity of individual tissues, parAbstract published in Advance ACS Abstracts, June 15, 1995. ticularly the lung, to NNK tumorigenesis is in part due Abbreviations: NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-l-b~to the efficient a-hydroxylation of NNK by these tissues tanone; NBzMA, N-nitrosobenzylmethylamine;NMPeA, N-nitrosomethylpentylamine; NNAL, 4-(methylnitrosamino)-l-(3-pyridyl)-l- (14-1 7). Two other factors which may contribute to this butanol; a-hydroxymethylNNK, 4-((hydroxymethyl)nitrosamino)-l-(3are the following: the relative capacity of different tissues pyridyll-l-butanone; a-hydroxymethylNNK-Gluc, p-O-[[N-nitroso-Nto detoxify the a-hydroxynitrosamine, and the preferen~4-oxo-4-~3-py~dyl)but-l-yl~aminolmethyl]-~-glucosiduronic acid; PB, phenobarbital; OPB, 4-oxo-l-(3-pyridyl)-l-butanone; NNAL-Gluc, p-0tial uptake of the active metabolite by the target tissue. ~4-~methylnitrosamino~-l-~3-pyridyl~but-l-yll-~glucosid~o~c acid; HPB, The formation of a glucuronide of the a-hydroxynitros4-hydroxy-l-(3-pyridyl)-l-butanone; "K-N-oxide, 4-(methylnitrosamiamine would initially detoxify it, but would also serve to no)-l-(N-oxy-3-pyridyl)-l-butanone; NNAL-N-oxide, 44methylnitrosamin0)-l-(N-0~-3-pyridyl)-l-b~k101; HPB-Gluc,p-0-[40~0-4-( 3-pyridyl)extend its lifetime. a-Hydroxynitrosamines have halfbut-l-yll-D-glucosiduronic acid; diol, 4-hydroxy-l-(3-pyridyl)-l-butanol; lives on the order of seconds (18). The glucuronide OPBA, 4-oxo-4-(3-pyridyl)butyricacid; HPBA, 4-hydroxy-4-(3-pyridyl)conjugate could serve as a stable transport form of the butyric acid; ESI,electrospray ionization.
Introduction
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Q893-228x/95/2708-Q772$09.QQIQ0 1995 American Chemical Society
Chem. Res. Toxicol., Vol. 8, No. 5, 1995 773
Glucuronidation of HydroxymethylNNK by Rats
NNAL-GIuc
1
-
NNK-N-oxide
-
&C' Hs NNK
-, C+H '3
0
-[
+nc:H*o
NNAL-N-oxide
0
E
whydroxymethyl NNK-GIUC
1
[
&'CH,Oti]
3HC'H& @
pGluc
a-hydroxymelhyl NNK
I
1 1
HPB-Glue
HPO
OPOA
HPBA
Figure 1. NNK metabolism to NNAL-Gluc, HPB-releasing glucuronides, a-hydroxylation products, and N-oxides. A small amount of HPBA is formed from OPB, but the majority, both in urine of NNK-treated rats and in cell culture, is from a-hydroxylation of NNAL. (UDPGT, uridine disphosphate glucuronosyltransferase;,B-Gluc, ,&glucuronidase.)
active metabolite which would travel from the tissue where it was formed to the target tissue. Wiessler and co-workers provided some evidence for the formation of glucuronides of the a-hydroxynitrosamines of N-nitrosobenzylmethylamine (NBzMA) and N-nitrosomethylpentylamine ( W e & 19,201. The only conjugate of a NNK metabolite that has been identified is a glucuronide of NNAL (Figure 11, which is present in the urine of rats, mice, and the patas monkey following NNK treatment (9, 21). NNAL-Gluc has also been quantitated in the urine of smokers, individuals exposed to sidestream smoke, and Sudanese snuff dippers (22-24). In the present study, we report the characterization of the glucuronide of a-hydroxymethylNNK (Figure 1). This glucuronide was present in the urine of rats treated with NNK and PB. It was also formed by hepatocytes from PB-treated rats during incubation with NNK. The experiments in this study were initiated to investigate the effect of inducers of cytochrome P450 on the formation of "K hemoglobin adducts in vivo and in vitro (25).The finding of a-hydroxymethylNNK-Gluc was unexpected, due to the unstable nature of the aglycon.
Materials and Methods Chemicals. [FI-~HINNK(2.4-2.8 CUmmol) and [n~ethyl-~HlNNK (0.825 Ci/mmol) were obtained from Chemsyn Laboratories (Lenexa, KS). Purity of both [5-3H]NNK and [methyl3H]NNK was greater than 98% as determined by radioflow HPLC. The NNK metabolite standards HPBA, OPBA, NNALN-oxide, NNK-N-oxide, diol, HPB, 6-hydroxyNNK, and NNAL (Figure 1) were synthesized as previously by the Organic Synthesis Facility of the American Health Foundation (26,27). [5-3H]NNAL was collected from the media of hepatocytes incubated with [5-3HINNK. [5-3HlNNAL-Gluc(1.34 nCUnmol)
was collected from the urine of a n NNK-treated patas monkey (9). /3-Glucuronidase, Type 1X-A, and D-saccharic acid 1,4lactone were obtained from Sigma Chemical Co. (St. Louis, MO). Williams media was obtained from Flow Laboratories (Rockville, MD). All other chemicals were analytical grade, and solvents were HPLC grade. Animal Treatments. Male F344 rats were purchased from Charles River (Kingston, NY,or Raleigh, NC). They had free access to water and NIH-07 diet. Three different animal treatments were used. One group of rats was treated for 4 days with PB (40 mgkg/day, ip) and then given a single ip injection of [5-3H]NNKor [methyL3H1NNK(100 pgkg, ip) 24 h following the last injection of PB. A second group of rats received [5-3HlNNK (100 pgkg, ip) only. After receiving NNK, rats in each of these groups were transferred to stainless steel metabolism cages, and a 24 h urine sample was collected with cooling by dry ice. Urine was kept frozen a t -80 "C until analysis for NNK metabolites. Rats which were used for hepatocyte isolation were either untreated or treated with PB (40 mgkg/day, ip) for 4 days. Analysis of Urine for NNK Metabolites. Twenty-four hour urine was filtered through Whatman No. 1 paper. The volume of the filtrate was increased to 10 mL with distilled H20, and then each sample was applied to a Centricon 30 concentrator (Amicon, Beverly, MA) to remove proteins. The filtrate was mixed with NNK metabolite standards and analyzed by reverse phase HPLC. NTVK Metabolism by Hepatocytes. Hepatocytes were isolated by collagenase perfusion as described previously (28, 29). Viability as measured by trypan blue exclusion was 8590%. The hepatocytes were plated a t a cell density of 3 x lo6 cells per culture dish (100 mm) in Williams media supplemented with 10%fetal calf serum. Dishes were placed in a n incubator with a humidified atmosphere of 95% air/5% C02 at 37 "C for 2-3 h until cells attached. Media was removed and replaced with Williams media which contained [VHINNK at concentrations of 1 pM (sp act. = 2.4 CUmmol) or 1 mM (sp act. = 0.1 CUmmol) or 1 mM unlabeled NNK. Eighteen hours following
774 Chem. Res. Toxicol., Vol. 8, No. 5, 1995
Murphy et al.
Table 1. Effect of PB Treatment on Urinary NNK Metabolites" % radioactivit9 untreated metaboliteC HPBA OPBA NNAL-N-oxide NNAL-Gluc NNK-N-oxide HPB 6-hydroxy-NNK NNAL NNK
-8-Gluc 16.0 f 1.6 37.8 f 1.5 11.4 f 0.9 0.59 f 0.23 11.7 f 0.7 10.05 1.0 & 0.08
3.33 f 0.33 0.8 5 0.17
PB treated +S-Gluc 15.2 f 1.6 33.4 f 1.1 10.6 f 0.9
