The Journal of Toxicological Sciences
Online ISSN : 1880-3989
Print ISSN : 0388-1350
ISSN-L : 0388-1350
Letter
4-(Hydroxymethylnitrosamino)-1-(3-pyridyl)-1-butanone glucuronide has the potential to form 2’-deoxyguanosine and N-acetylcysteine adducts
Takahito NishiyamaNahoko HayashiHiromi YanagitaTomokazu OhnumaKenichiro OguraAkira Hiratsuka
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2019 Volume 44 Issue 10 Pages 693-699

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Abstract

Cigarette smoking is a risk factor for the development of various cancers, such as lung, nasal, liver and bladder cancers. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific nitrosamine, is implicated in human lung cancer. NNK-induced DNA adducts are found in target tissues for NNK carcinogenesis. NNK is activated by cytochrome P450 dependent α-hydroxylation at either the methylene carbon or methyl carbon adjacent to the N-nitroso group. The former leads to the formation of the methylating agent, and the latter produce the pyridyloxobutylating agent. NNK and some of its metabolites are further metabolized by UDP-glucuronosyltransferases (UGTs). Glucuronides generally are much less active than the parent aglycon therefore the glucuronides of NNK-related metabolites are thought to be inactive. However, 4-(hydroxymethylnitrosamino)-1-(3-pyridyl)-1-butanone glucuronide (HO-methyl NNK glucuronide) can be transported to the target organs of NNK carcinogenesis where subsequent hydrolysis causes the release of the reactive intermediate. Regeneration of HO-methyl NNK could play an important role in the tissue-specific carcinogenicity of NNK. In the present study, we investigated the reactivity of HO-methyl NNK glucuronide toward 2’-deoxyguanosine (dGuo) and N-acetylcysteine (NAC; used as a models for thiol groups on proteins). The reaction mixtures of HO-methyl NNK glucuronide and dGuo or NAC were analyzed by LCMS-IT-TOF-MS. We also employed 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone, a pyridyloxobutylating agent, to confirm the formation of pyridyloxobutylated adducts. Thus, we determined the production of pyridyloxobutylated dGuo and NAC adducts. Our results suggest HO-methyl NNK glucuronide could generate a reactive intermediate in the tissues and then form adducts with proteins and DNA.

INTRODUCTION

4-(Methylnitrosamino)-1-(3-pyridine)-1-butanone (NNK) is a nicotine-derived carcinogen present in tobacco smoke that is one of the tobacco-specific nitrosamines. NNK is classified as a group 1 compound, which are considered to be “carcinogenic to humans”, according to the International Agency for Research on Cancer (IARC) monograph (IARC, 2007). However, NNK requires metabolic activation by cytochrome P450 mediated α-hydroxylation to exhibit its carcinogenic properties. Activation of NNK proceeds by α-hydroxylation of either the α-methylene or methyl carbon adjacent to the N-nitroso group, producing reactive intermediates. These intermediates spontaneously decompose and have the potential to alkylate DNA. When the α-methylene adjacent to the N-nitroso group is hydroxylated the metabolically reactive intermediate 4-(hydroxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (HO-methyl NNK) is formed. HO-Methyl NNK spontaneously decomposes to generate 4-oxo-4-(3-pyridyl)-1-butanediazohydroxide. This reactive intermediate is believed to act as a pyridyloxobutylating agent to both DNA and proteins. Indeed, methylated and pyridyloxobutylated hemoglobin adducts are useful as biomarkers of exposure to tobacco-specific nitrosamine (Hecht, 1998).

NNK and some of its metabolites are glucuronidated by UDP-glucuronosyltransferases (UGTs) and are excreted as detoxified metabolites. Until now, the formation of four types of NNK-related glucuronides has been reported (Morse et al., 1990; Hecht et al., 1993; Wiener et al., 2004). Among them, HO-methyl NNK glucuronide is an interesting metabolite because it is a glucuronide of a metabolically reactive intermediate that is extremely unstable compared to the other aglycones. Specifically, HO-methyl NNK glucuronide is transported to the target organ of NNK carcinogenesis where the glucuronide is hydrolyzed by β-glucuronidase in the tissue to be reactivated as HO-methyl NNK (Fig. 1). It is believed that regeneration of HO-methyl NNK in tissues results in the formation of DNA adducts and may be involved in tissue-specific carcinogenesis of NNK (Murphy et al., 1995). In our laboratory, we have investigated whether HO-methyl NNK glucuronide is likely to be a detoxified metabolite in vivo or is involved in the tissue-specific carcinogenic effects of NNK. We previously reported that HO-methyl NNK glucuronide is present in the target organ of NNK carcinogenesis (Nishiyama et al., 2014a, 2014b). Furthermore, we examined the stability of HO-methyl NNK glucuronide in mouse liver homogenate and revealed that it is rapidly degraded at pH 6.2.

Fig. 1

HO-Methyl NNK glucuronide, a stable form of a reactive intermediate, could be distributed to target organs associated with NNK-induced carcinogenesis. The glucuronide may then be hydrolyzed to generate a reactive intermediate. The regenerated HO-methyl NNK could covalently bind to DNA or protein.

In the present study we aimed to clarify the mechanism of tissue-specific carcinogenicity of NNK. Specifically, we investigated whether HO-methyl NNK glucuronide reacts with 2’-deoxyguanosine (dGuo) to form a pyridyloxobutyl-dGuo adduct. Furthermore, we evaluated the reactivity of HO-methyl NNK glucuronide with thiol groups on proteins by investigating adduct formation between HO-methyl NNK glucuronide and N-acetylcysteine (NAC).

MATERIALS AND METHODS

Materials

Oasis HLB (1 mL (30 mg) and 3 mL (60 mg)) cartridges were purchased from Waters (Milford, MA, USA). dGuo, β-glucuronidase Type IX-A and porcine liver esterase were purchased from Sigma-Aldrich (St Louis, MO, USA). NAC and trichloroacetic acid (TCA) was obtained from Kanto Chemical Co., Inc. (Tokyo, Japan). 4-(Acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (HO-methyl NNK Ac) and NNK were purchased from Toronto Research Chemicals, Inc. (North York, ON, Canada). All other reagents were of the highest grade commercially available.

Purification of HO-methyl NNK glucuronide

Isolation of HO-methyl NNK glucuronide from urine was performed as described previously (Nishiyama et al., 2014b).

Reaction of HO-methyl NNK glucuronide with dGuo

HO-Methyl NNK glucuronide (0.5 µmol) was allowed to react with dGuo (20 µmol) in 2 mL of 100 mM phosphate buffer (pH 5.0) in the presence of β-glucuronidase (2,000 units) at 37°C for 1.5 hr. The reaction conditions were selected taking into account the optimum pH of β-glucuronidase activity and the reactivity of dGuo. The reaction was terminated by the addition of 2 mL of TCA (10% w/v) and centrifuged at 10,000 x g for 15 min. The resulting supernatant was loaded onto an Oasis HLB 3 mL cartridge. The cartridge was washed with 3 mL of water and then eluted with 3 mL of methanol. The eluate was concentrated to dryness under a gentle stream of nitrogen at a temperature of 40°C. The dry residue was dissolved in 100 µL of the mobile phase. A 1 µL aliquot was analyzed by HPLC.

Reaction of HO-methyl NNK glucuronide with NAC

HO-Methyl NNK glucuronide (0.5 µmol) was allowed to react with NAC (20 µmol) in 2 mL of 10 mM phosphate buffer (pH 6.2) in the presence of mouse liver homogenate (5 mg/mL) at 37°C for 1.5 hr. The reaction conditions were selected taking into account the optimum pH on the degradation of HO-methyl NNK glucuronide in mouse liver homogenate, as previously reported (Nishiyama et al., 2014b). The reaction was terminated by the addition of 2 mL of TCA (10% w/v) and centrifuged at 10,000 x g for 15 min. The resulting supernatant was loaded onto an Oasis HLB 3 mL cartridge. Solid-phase extraction was performed as described above.

Reaction of HO-methyl NNK Ac with dGuo

HO-Methyl NNK Ac (5 µmol) was allowed to react with dGuo (1 µmol) in 100 µL of 100 mM phosphate buffer (pH 5.0) in the presence of esterase (27 units) at 37°C for 1 hr. The reaction was terminated by the addition of 100 µL of TCA (10% w/v) and centrifuged at 10,000 x g for 15 min. The resulting supernatant was loaded onto an Oasis HLB 1 mL cartridge. The cartridge was washed with 1 mL of water and then eluted with 1 mL of methanol. The eluate was concentrated to dryness under a gentle stream of nitrogen at a temperature of 40°C. The dry residue was dissolved in 100 µL of the mobile phase. A 1 µL aliquot was analyzed by HPLC.

Reaction of HO-methyl NNK Ac with NAC

HO-Methyl NNK Ac (5 µmol) was allowed to react with NAC (1 µmol) in 100 µL of 10 mM phosphate buffer (pH 6.2) in the presence of esterase (27 units) at 37°C for 1 hr. The reaction was terminated by the addition of 100 µL of TCA (10% w/v) and centrifuged at 10,000 x g for 15 min. The resulting supernatant was loaded onto an Oasis HLB 1 mL cartridge. Solid-phase extraction was performed as described in the section for reaction of HO-methyl NNK Ac with dGuo.

LCMS-IT-TOF-MS (LC/MS/MS) analysis

Samples (1 µL) were analyzed using a Shimadzu Prominence (Shimadzu Co, Kyoto, Japan) interfaced with a quadrupole ion trap time-of-flight mass spectrometer (LCMS-IT-TOF, Shimadzu Co) equipped with an ESI source. Separation was achieved using a Capcellpak (2.0 x 50 mm, 5 mm; Osaka Soda Co. Ltd., Osaka, Japan). For determination of pyridyloxobutyl-dGuo adducts, the mobile phase consisted of 10 mM ammonium acetate buffer (pH 5.0) and acetonitrile. Elution was performed on a linear gradient from 1 to 20% acetonitrile over 19 min at a flow rate of 0.2 mL/min. A triple quadrupole mass spectrometer fitted with an ESI source operated in positive ion mode was used. For determination of pyridyloxobutyl-NAC adducts, the mobile phase consisted of water containing 0.1% formic acid and acetonitrile. Elution was performed on a linear gradient from 5 to 25% acetonitrile over 20 min at a flow rate of 0.2 mL/min. A triple quadrupole mass spectrometer fitted with an ESI source operated in positive ion mode was used.

RESULTS AND DISCUSSION

HO-Methyl NNK glucuronide and dGuo were incubated at 37ºC in the presence of β-glucuronidase to investigate the possible formation of pyridyloxobutyl-dGuo adducts. The reaction products were subsequently identified by LCMS-IT-TOF-MS. The precursor m/z 415.17 and product m/z 299.12 in positive ion mode was monitored. The m/z 415.17 was consistent with the protonated molecular ion of pyridyloxobutyl-dGuo. The LC/MS/MS chromatogram showed a peak with a retention time at 9.60 min (Fig. 2A). This peak (9.6 min) was not detected in the absence of β-glucuronidase. MS/MS fragmentation resulted in the formation of a characteristic fragment ion at m/z 299.12, corresponding to the parent ion with loss of a 2’-deoxyribose moiety (Fig. 2B). Moreover, the MS/MS of 299.12 is explicable by formation of the characteristic fragment ions at m/z 148.07 [3-Pyr(CO)(CH2)3]+ and 152.05 (guanine+H)+ (Fig. 2C).

Fig. 2

Liquid chromatography-mass spectrometry analysis of the reaction mixture after incubation of HO-methyl NNK glucuronide and dGuo in the presence of β-glucuronidase. In each panel, A is an LC/MS/MS chromatogram of 415.17→299.12; B is the corresponding MS spectrum; C is the daughter ion spectrum of m/z 299.12.

Formation of pyridyloxobutyl-dGuo adducts using HO-methyl NNK Ac has already been reported in detail (Hecht et al., 1986; Wang et al., 2003). These studies identified four isomers of pyridyloxobutyl-dGuo adducts (m/z 415; O6-pyridyloxobutyl-dGuo, 7-pyridyloxobutyl-dGuo, N2-pyridyloxobutyl-dGuo, N2-pyridyloxobut-2-yl-dGuo). Therefore, we employed HO-methyl NNK Ac as a pyridyloxobutylating agent, which was incubated with dGuo at 37ºC in the presence of esterase. The major peak appeared at 9.57 min, and the retention time was the same as that of the adduct formed between HO-methyl NNK glucuronide and dGuo (Fig. 3A). Furthermore, a fragment ion of m/z 299.12 was detected in the MS/MS spectrum (Fig. 3B). The daughter ion peaks at m/z 148.08 and 152.06 were also detected in MS/MS of m/z 299.12 (Fig. 3C). The peak patterns and retention times obtained from LCMS-IT-TOF-MS analysis were identical to those of the dGuo adduct of HO-methyl NNK glucuronide. From these results, we identified the 9.6 min peak as the pyridyloxobutyl-dGuo adduct.

Fig. 3

Liquid chromatography-mass spectrometry analysis of the reaction mixture after incubation of HO-methyl NNK Ac and dGuo in the presence of esterase. In each panel, A is an LC/MS/MS chromatogram of 415.17→299.12; B is the corresponding MS spectrum; C is the daughter ion spectrum of m/z 299.12.

When HO-methyl NNK glucuronide and dGuo were incubated in the presence of mouse bladder homogenate, the peak patterns and retention time obtained from LCMS-IT-TOF-MS analysis was identical to that of the pyridyloxobutyl-dGuo adducts (results not shown). These results suggested that HO-methyl NNK glucuronide may be hydrolyzed by β-glucuronidase in bladder tissue, and reactivated HO-methyl NNK could subsequently form adducts with DNA.

HO-Methyl NNK glucuronide and NAC were incubated at 37ºC in the presence of mouse liver homogenate. We then attempted to detect the formation of pyridyloxobutyl-NAC adducts. The LC/MS mass spectrum showed a protonated molecular ion [M+H]+ (m/z 311.14 for pyridyloxobutyl-NAC) peak at retention time 9.16 min, consistent with the molecular ion of pyridyloxobutyl-NAC (Fig. 4A). This peak (9.16 min) was not detected in the absence of mouse liver homogenate. Furthermore, MS/MS of m/z 311.4 produced daughter ions at m/z 130.07 and m/z 148.08 [3-Pyr(CO)(CH2)3]+ (Fig. 4B). Therefore, we identified the peak at 9.16 min as a pyridyloxobutyl-NAC adduct. In addition, HO-methyl NNK Ac and NAC were incubated at 37ºC in the presence of an esterase and adduct formation between HO-methyl NNK Ac and NAC examined. When the ion at m/z 311.14 was monitored, a peak appeared at a retention time of 9.11 min. The retention time of this peak was the same as that of the NAC adduct formed by HO-methyl NNK glucuronide. Furthermore, fragment ion peaks at m/z 130.7 and 148.08 were also detected in the MS/MS spectrum. These fragment patterns were similar to that of the NAC adduct of HO-methyl NNK glucuronide. Structural characterization of NAC adducts has already been reported in detail (Carmella et al., 1990). The report by Carmella et al. describes five NAC adduct isomers with a m/z 311 (including stereoisomers).

Fig. 4

Extracted ion chromatogram (m/z 311.4) (A) and MS/MS spectrum of NAC adduct (B) after incubation of HO-methyl NNK glucuronide with NAC in the presence of mouse liver homogenate.

In this study, we show for the first time the possibility that HO-methyl NNK glucuronide, which appears to be a detoxified metabolite, may be subject to β-glucuronidase cleavage to generate a reactive intermediate capable of forming adducts with both DNA and proteins. Studies on the relationship between urinary pH, cigarette smoking, and bladder cancer risk have been reported previously (Alguacil et al., 2011). These investigations suggested that urine pH may modify the impact of tobacco use on the risk of bladder cancer. Alguacil et al. (2011) also point out that this conclusion is consistent with experimental data showing that acidic urine can result in cleavage of acid-labile glucuronides of carcinogenic aromatic amines (Kadlubar et al., 1977). Thus, during excretion in acidic urine HO-methyl NNK glucuronide may have the potential to form adducts with proteins and DNA. Consequently, the relationship between the results of our study and bladder cancer in smokers is of interest.

Conflict of interest

The authors declare that there is no conflict of interest.

REFERENCES
 
© 2019 The Japanese Society of Toxicology
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